Methods and compositions for stem cell self-renewal

ABSTRACT

The present invention relates to methods for expanding a stem cell population. More particularly, the invention relates, inter alia, to methods and compositions for expanding a stem cell population, particularly a hematopoietic stem cell population.

RELATED APPLICATIONS

This application claims benefit of U.S. Patent Application Ser. No.60/926,065, filed on Apr. 23, 2007, and U.S. Patent Application Ser. No.61/066,693, filed on Feb. 22, 2008, and which applications areincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for expandinga stem cell population, particularly an hematopoietic stem cellpopulation.

BACKGROUND OF THE INVENTION

Hematopoietic stem cells (HSCs) are clonogenic cells, which possess theproperties of both self-renewal (expansion) and multilineage potentialgiving rise to all types of mature blood cells. HSCs are responsible forhematopoiesis and undergo proliferation and differentiation to producemature blood cells of various lineages while still maintaining theircapacity for self-renewal. The ability to self-renew maintains the HSCpopulation for the lifespan of an animal and also allows HSCs torepopulate the bone marrow of lethally irradiated congenic hosts.

Early HSC development displays a hierarchical arrangement, starting fromlong-term (LT-) HSCs, which have extensive self-renewal capability,followed by the expansion state, which corresponds to short-term (ST-)HSCs (having limited self-renewal ability) and proliferative multipotentprogenitors (MPPs) (having multipotent potential but no self-renewalcapability). MPP is also a stage of priming or preparation fordifferentiation. An MPP differentiates and commits to become either acommon lymphoid progenitor (CLP), which gives rise to all the lymphoidlineages, or a common myeloid progenitor (CMP), which produces all themyeloid lineages. During this process, the more primitive populationgives rise to a less primitive population of cells, which is unable togive rise to a more primitive population of cells. The intrinsic geneticprograms that control these processes including the multipotential,self-renewal, and activation (or transient amplification) of HSCs, andlineage commitment from MPP to CLP or CMP, remain largely unknown.

To sustain constant generation of blood cells for the lifetime of anindividual, HSCs located in bone marrow niches (Zhang, J. et al. Nature425, 836-841, 2003; Calvi, L. M. et al. Nature 425, 841-846, 2003; Kiel,M. J., et al. Cell 121, 1109-1121, 2005; Arai, F. et al. Cell 118,149-161, 2004) must achieve a balance between quiescence and activationso that immediate demands for hematopoiesis are fulfilled, whilelong-term stem cell maintenance is also assured. In adults, homeostasisbetween the quiescent and activated states of stem cells is important toprotect HSCs from losing their potential for self-renewal and, at thesame time, support ongoing tissue regeneration (Li, L. and Xie, T. Annu.Rev. Cell. Dev. Biol. 21, 605-631, 2005). Over-activation and expansionof stem cells risks both eventual depletion of the stem cell populationand a predisposition to tumorigenesis. Although some factors importantfor stem cell activation have been identified (Heissig, B. et al. Cell109, 625-637, 2002), the molecular events governing the transitionbetween quiescence and activation are poorly understood.

Phosphatase and tensin homolog (PTEN) functions as a negative regulatorof the PI3K/Akt pathway, which plays crucial roles in cellproliferation, survival, differentiation, and migration (Stiles, B. etal. Dev. Biol. 273, 175-184, 2004). The PTEN tumor suppressor iscommonly mutated in tumors, including those associated with lymphoidneoplasms, which feature deregulated hematopoiesis (Mutter, G. L. Am. J.Pathol. 158, 1895-1898, 2001; Suzuki, a. et al. Immunity 14, 523-534,2001). PTEN-deficiency has been associated with expansion of neural andembryonic stem cell populations (Groszer, M. et al. Science 294,2186-2189, 2001; Kimura, T. et al. Development 130, 1691-1700, 2003).But, the role of PTEN in stem cells and tumorigenesis and the recurrenceof tumors heretofore has been not understood.

PTEN functions as an antagonist of phosphatidyl inositol 3-kinase (PI3K)(Maehama T & Dixon J E. J Biol. Chem. 273:13375-13378. 1998). The serinekinase Akt is downstream of the PI3K signal (Cross D A, Alessi D R,Cohen P et al. Nature 378:785-789 1995). PTEN has been shown to inhibitAkt and thereby inhibit the nuclear accumulation of β-catenin (Persad Set al. J. Cell Biol. 153:1161-1174 2001).

Akt has a broad range of effects. Its major function is to provide asurvival signal and to block apoptosis, complementary to its regulationof Jβ-catenin function. (Song, G. et al., J. Cell. Mol. Med., 9(1):59-71, 2005) Akt acts through a number of proteins, including mammaliantarget of rapamycin (mTOR), the Forkhead family of transcription factors(FoxO), BAD, caspase 9, murine double minute 2 (Mdm2).

Akt can directly and indirectly activate the serine/threonine kinasemTOR, which activates protein translation through a signaling cascade.(LoPiccolo, J., et al., Anti-Cancer Drugs, 18:861-874, 2007). Indirectactivation occurs through tuberous sclerosis complex-2 (TSC2), which,when in the unphosphorylated state, forms a complex with tuberoussclerosis complex-1 (TSC1, also known as hamartin). This complexpromotes the GTPase activity of Ras homolog enriched in brain (RHEB),which in turn, acts to down-regulate mTOR activity. Upon phosphorylationby Akt, however, the ability of the TSC1-TSC2 complex to promote RHEB'sGTPase activity is inhibited, and therefore, mTOR′ s activity ispromoted. (Cully, M. et al., Nat. Rev. Cancer, 6:184-192, 2006). mTORcan also form a complex with Rictor, and this complex can providepositive feedback on the Akt signaling cascade by phosphorylating andactivating Akt. (Sarbassov, D. D., et al., Science, 307: 1098-1101,2005).

Akt also regulates cell survival through transcriptional factors,including FoxO. Akt's phosphorylation of FoxO inhibits FoxO, resultingin inhibition of transcription of several proapoptotic genes, such asFas-L, IGFBP1 and Bim. (Datta, S. R., et al., Cell, 91:231-241, 1997;Nicholson, K. M., et al., Cell Signal, 14:381-395, 2002).

One of the down-stream targets of FoxO is p27 (Kip1), a potent inhibitorof cyclin E/cdk2 complexes. (Wu, H. et al., Oncogene, 22: 3113-3122,2003). FoxO factors induce expression of p27, which can bind to cyclinE/cdk2 complexes and inhibit their activity, resulting in a block incellular proliferation. (Burgering, B. M. T. & Medema, R. H., J.Leukocyte Biol., 73:689-701, 2003). In addition, Akt itself can alsodirectly phosphorylate p27 on T157, resulting in the redistribution ofp27 from the nucleus to the cytoplasm, away from cyclin E/cdk2complexes. (Id.) Phosphorylation of p27 on T198 was critical for thebinding of p27 to 14-3-3 proteins, and through this pathway, Akt maydirectly promote p27's degradation. (Fujita, N., et al., J. Biol. Chem.,277(32): 28706-28713, 2002).

Another one of the targets of Akt in promoting cell survival is BAD, amember of the Bcl-2 family of proteins. In the absence of Aktphosphorylation, BAD forms a complex with Bcl-2 or Bcl-X on themitochondrial membrane and inhibits the anti-apoptotic potential ofBcl-2 and Bcl-X. (Song, G. et al., J. Cell. Mol. Med., 9(1): 59-71,2005) Akt phosphorylates BAD on Serine 136, thus releasing BAD from theBcl-2/Bcl-X complex. (Song, G. et al., J. Cell. Mol. Med., 9(1): 59-71,2005; Datta, S. R., et al., Genes Dev., 13:2905-2927, 1999). Therefore,Akt suppresses BAD-mediated apoptosis and promotes cell survival.

Furthermore, by phosphorylation of pro-caspase-9 at Serine 196, Aktinhibits proteolytic processing of pro-caspase-9 to the active form,caspase-9, which is an initiator and an effecter of apoptosis (Cardoneet al., 1998, Science, 282: 1318-1320, Donepudi, M. & Grutter, M. G.,Biophys. Chem., 145-152, 2002).

Additionally, Akt regulates cell survival via the Mdm2/p53 pathway. Aktcan activate Mdm2 by direct phosphorylation, thereby inducing thenuclear import of Mdm2 or the up-regulation of Mdm2'subiquitin ligaseactivity. (Mayo L. D., Donner D. B., 2001, Proc. Natl., Acad. Sci. USA98:11598-11603; Gottlieb T. M. et al, Ocogene, 21: 1299-1303, 2002).Mdm2 negatively regulates the p53 protein, which may induce cell deathin response to stresses (Oren M., Cell Death Differ., 10:431-442, 2003),by targeting p53 for ubiquitin-mediated proteolysis (Haupt, Y. et al.,1997, Nature 387: 296-299) or by binding to the transactivation domainof p53, thereby inhibiting p53-mediated gene regulation. (Momand, J. etal., Cell, 69: 1237-1245, 1992) One of the down-stream targets of p53 isthe p21 (CIP1/WAF1) gene. The p53 gene product binds to a site located2.4 kb upstream of the p21 coding sequence, and this binding siteconfers p53-dependent transcriptional regulation. (El-Deiry, W. S., etal., Cell, 75: 817-825, 1993) Thus, down-regulation of p53 alsodown-regulates the transcription of p21.

PTEN not only regulates p53 protein through antagonizing the Akt-Mdm2pathway, it can also directly regulate p53. First, PTEN can enhance p53transactivation in a phosphatase-independent manner (Tang, Y. & Eng C.,Cancer Research, 66: 736-742, 2006). Second, PTEN forms a complex withp300 in the nucleus and plays a role in maintenance of high p53acetylation, which is the activated form of p53. (Li A. et al.,Molecular Cell, 23 (4): 575-587, 2006). In turn, p53 may also activatethe transcription of PTEN. (Cully, M. et al., Nat. Rev. Cancer,6:184-192, 2006).

Canonical signals in the Wnt pathway are involved in stem cellproliferation. (Kim, L. & Kimmel, A. R. Current Drug Targets7:1411-1419, 2006). Glycogen synthase kinase 3 beta (GSK-3β) is a partof the Wnt signaling pathway, and its primary substrate is β-catenin.(Hagen, T et al., J. Biochem. 277(26):23330-23335). In the absence ofcanonical Wnt signaling, GSK-3β binds to β-catenin and phosphorylatesβ-catenin, thereby targeting β-catenin for ubiquitination and followedby proteosome-mediated degradation, which is mediated by AdenomatousPolyposis Coli (APC). (Id., Moon, R. T. et al., Science 296:1644-1646.2002). Canonical Wnt signals induce the release of β-catenin fromGSK-3β, thereby activating β-catenin. (Katoh, M & Katoh, M. Cancer BiolTher. 5(9):1059-64, 2006). β-catenin then localizes to the nucleus,where it activates gene transcription. (Id.).

In sum, individually it was known or suggested that modulating GSK-3βand PTEN could promote stem cell, e.g., HSC, proliferation. But, suchknowledge and speculation was insufficient to provide a clinicallyuseful approach to expanding a stem cell population for subsequenttransplantation and reconstitution of the engrafted cell lineage.

In view of the foregoing, it would be advantageous to elucidate theinteraction between Wnt and PTEN signaling pathways and to provide newinsights into molecular regulation of stem cell proliferation anddifferentiation. It would also be advantageous to use such insights toprovide new methods and compositions for expanding stem cells in vivoand ex vivo, which stem cells would be of the kind and quantitysufficient to transplant into a suitable recipient.

SUMMARY OF THE INVENTION

Thus, one embodiment of the invention is a method for expanding apopulation of stem cells obtained from a tissue selected from the groupconsisting of peripheral blood, cord blood, and bone marrow. This methodcomprises modulating a PTEN pathway and a Wnt pathway in the populationof stem cells to expand the number of stem cells.

Another embodiment of the invention is a method for ex vivo expansion ofa substantially undifferentiated stem cell population. This methodcomprises modulating a PTEN pathway and a Wnt pathway in theundifferentiated stem cell population to expand the number ofundifferentiated stem cells without significant differentiation of thestem cell population.

Yet another embodiment of the invention is a method for ex vivoexpansion of an hematopoietic stem cell (HSC) population obtained from atissue selected from the group consisting of peripheral blood, cordblood, and bone marrow. This method comprises modulating a PTEN pathwayand a Wnt pathway in the HSC population to expand the HSC population toa sufficient quantity while maintaining a multilineage differentiationpotential in the HSC population, which is sufficient for subsequenttransplantation into a patient in need thereof.

Another embodiment of the invention is an expanded, substantiallyundifferentiated stem cell population made by a method of the presentinvention. In a related embodiment, the invention is an expanded HSCpopulation made by a method of the present invention.

An additional embodiment is a method for ex vivo expansion ofhematopoietic stem cells (HSCs) by at least 40-fold, the expanded HSCsbeing competent to reconstitute an HSC lineage upon transplantation intoa mammalian patient in need thereof. This method comprises culturing apopulation of HSCs in a suitable culture medium comprising a PTENinhibitor and a GSK-3β inhibitor.

A further embodiment of the invention is a kit for expanding anhematopoietic stem cell (HSC) population for subsequent transplantationinto a patient in need thereof. The kit comprises a PTEN inhibitor, aGSK-3β inhibitor, and instructions for the use of the inhibitors.

An additional embodiment is a media for carrying out ex vivo expansionof a stem cell population. The media comprises a fluid media suitablefor maintaining viable stem cells and PTEN and GSK-3β inhibitors presentin the media at concentrations sufficient to enable expansion of thestem cell population while maintaining a multilineage differentiationpotential in the stem cells.

Yet another embodiment is a method for administering an hematopoieticstem cell (HSC) to a patient in need thereof. This method comprises (a)culturing, in a suitable culture media, a sample containing an HSCpopulation in the presence of a modulator of a molecule in the PTENpathway and a modulator of a molecule in the Wnt pathway for a period oftime sufficient to expand the number of HSCs in the sample to a numbersufficient to transplant into the patient; (b) removing from the culturethe PTEN and Wnt pathway modulators; and (c) administering the HSCs tothe patient.

A further embodiment of the invention is a method for reconstitutingbone marrow in a patient in need thereof. This method comprises: (a)culturing, in a suitable culture media, a sample containing an HSCpopulation in the presence of a modulator of a molecule in the PTENpathway and a modulator of a molecule in the Wnt pathway for a period oftime sufficient to expand the number of HSCs in the sample to a numbersufficient to transplant into the patient; (b) removing from the culturethe PTEN and Wnt pathway modulators; and (c) administering the HSCs tothe patient.

Another embodiment is a method for expanding a population ofhematopoietic stem cells (HSCs). This method comprises culturing apopulation of HSCs under conditions sufficient to result in an expansionof the HSC population by at least 40-fold, wherein the expandedpopulation of HSCs is suitable for transplantation into a mammal in needthereof.

Yet another embodiment is a method for treating a patient in need of atransplant selected from the group consisting of a bone marrowtransplant, a peripheral blood transplant, and an umbilical cord bloodtransplant. This method comprises administering to the patient apopulation of HSCs obtained by a method of the present invention.

A further embodiment is a method for expanding a population ofhematopoietic stem cells (HSCs) comprising: (a) obtaining from a mammala tissue sample comprising an HSC population; (b) expanding, in vitro,the HSC population from the sample, wherein: (i) the HSC populationexpands by at least 40-fold; and (ii) the expanded HSC population hasthe ability to reconstitute an hematopoietic lineage for at least4-weeks after transplantation into a recipient.

An additional embodiment is a method for reconstituting an hematopoieticstem cell lineage in a recipient in need thereof. This method comprises:(a) obtaining from a mammal a tissue sample comprising an HSCpopulation; (b) expanding, in vitro, the HSC population from the sample,wherein: (i) the HSC population expands by at least 40-fold; and (ii)the expanded HSC population has the ability to reconstitute anhematopoietic lineage for at least 4-weeks after transplantation into arecipient in need thereof; and (c) transplanting the expanded HSCpopulation into a recipient in need thereof.

A further embodiment of the invention is a method for expanding ahematopoietic stem cell population in a mammal in need of suchexpansion. This method comprises administering to the mammal atherapeutically effective amount of a modulator of Wnt and Akt for aperiod of time sufficient to expand the HSC population by at least40-fold with HSCs that possess the ability to reconstitute anhematopoietic lineage in the mammal.

These and other aspects of the invention are further disclosed in thedetailed description and examples which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-K are a series of bar graphs and fluorescence activated cellscanning (“FACS”) analyses that collectively show that loss of PTEN withconstitutively active β-catenin leads to hematopoietic stem cell (HSC)expansion with loss of early hematopoietic progenitors.

FIG. 1A is two bar graphs showing the absolute numbers (per femur+tibia)of lineage negative, Sca-1⁺ Kit⁺ (LSK) cells in Scl-Cre negative controland Scl-Cre⁺ PTEN with constitutively activated β-catenin (Pten:Ctnnb1)double mutant and each single mutant bone marrow (top) and spleen(bottom) as determined by FACS analysis. (Harada, N., et al., Embo J,18(21): 5931-42 1999. Yilmaz, O. H., et al., Nature, 441:475-82 2006.Zhang, J., et al., Nature, 441(7092): 518-22 2006.) Mice are at 10 dayspost-induction of Tamoxifen. Reduction of LSK cells in double mutantbone marrow with expansion in the spleen is indicative of mobilizationfrom bone marrow to spleen. Scl-Cre is an HSC-specific Tamoxifeninducible Cre-recombinase used to achieve conditional knockout of LoxPflanked (floxed) Pten and Ctnnb1 alleles. (Gothert, J. R., et al.,Blood, 105(7): 2724-2732, 2005.)

FIGS. 1B-1E show representative results of FACS analysis of lineagenegative, Sca-1⁺ Kit⁺ (LSK) cells in Scl-Cre negative control (B and D)and Scl-Cre⁺ PTEN with constitutively activated β-catenin (Pten:Ctnnb1)double mutant (C and E) bone marrow and spleen as indicated. Boxes onthe left show Sca-1⁺ Kit⁺ (early hematopoietic progenitor cells), andboxes on the right show Sca-1⁺ Kit⁺ (LSK) cells. Cells were pre-gated onlive, lineage negative cells. Cells were collected from mice at 6 weekspost-induction of Tamoxifen.

FIGS. 1F and G are bar graphs showing the absolute number of LSK cellsper femur and tibia in control, Ctnnb1, Pten, and Pten:Ctnnb1 doublemutant bone marrow (F) and spleen (G) at 6 weeks post-induction. Whilethe percentage of LSKs is increased in double mutants (see FIG. 1C), lowcellularity of bone marrow from double mutants yields only moderatelyincreased absolute numbers compared to control.

FIGS. 1H and I are bar graphs and FACS analysis, respectively, ofpercentage of LSK cells, which are Flk2⁻ (indicating long-termreconstituting (LT)-HSCs) in control, Ctnnb1, Pten, and Pten:Ctnnb1mutant bone marrow at 6 weeks post-induction. Ctnnb1 single mutants arenot significantly different from controls at this time point (data notshown). Boxes in FIG. 1I indicate Flk2⁻ (LT HSC) cells.

FIG. 1J is a set of FACS analyses of CD45 in leukemic Pten:Ctnnb1 mutantbone marrow. CD45 (high) blast crisis cells are indicated (blue box,left panel). No blast cell population is observed in control or Ctnnb1mutants while a minor population has been observed in 1 of 1 Pten singlemutant mice at 6 weeks post-induction (data not shown). The right panelshows LSK analysis of leukemic Pten:Ctnnb1 mutant mouse bone marrow.Note the conversion to blast cells (lower left) with only a remnant LSKpopulation (compare to FIG. 1C).

FIG. 1K is a bar graph showing early hematopoietic progenitors definedby FACS analysis in control, Ctnnb1, Pten, and Pten:Ctnnb1 double mutantbone marrow. Common myeloid progenitor (CMP); granulocyte-monocyteprogenitor (GMP); megakaryocyte-erythrocyte progenitor (MEP); and commonlymphoid progenitor (CLP).

FIGS. 2A-J are a series of photographs, bar graphs, and FACS analysesthat collectively show that double mutant HSCs expand dramatically invitro and in vivo but fail to differentiate.

FIG. 2A is a series of photographs showing 100 LSK cells isolated fromcontrol, active β-catenin (Ctnnb1), Pten mutant, and double mutant(Pten:Ctnnb1) mice after 10 days in culture (original magnification100×). Cell numbers are not dramatically increased from 100 seeded LSKsin control while Ctnnb1 single mutant LSKs do not survive. In contrast,Pten single mutant LSKs exhibit greater proliferation but appear moreheterogeneous indicating more significant differentiation. The greatestand most homogeneous expansion occurs from Pten:Ctnnb1 double mutantLSKs.

FIG. 2B is a set of photographs showing LSK cells from Pten andPten:Ctnnb1 mutants at 34 days culture (original magnification 200×).(Note: wild-type control cultures do not expand beyond 4 weeks; Ctnnb1mutant cultures do not survive beyond 10 days.) Pten mutant HSC culturesappear more heterogeneous with significant cell clumping and moreirregular cell morphology. Also note the spindle-shaped adherent cells(arrows) showing differentiation. In contrast, double mutant HSCcultures exhibit consistent morphology. Therefore, while Pten singlemutant LSKs survive and expand, they have undergone more significantdifferentiation than the much more homogeneous Pten:Ctnnb1 double mutantLSKs.

FIGS. 2C and D are bar graphs showing the results of an expansionexperiment. Pten and Pten:Ctnnb1 LSK seven week cultures were countedand analyzed by FACS for maintenance of the LSK phenotype (wild-typecontrol and Ctnnb1 cultures did not survive this long in vitro). Doublemutant LSKs undergo >1,200 fold expansion vs. 50 fold for Pten singlemutant LSKs. LSK purity of cultures is significantly greater inPten:Ctnnb1 cultures maintaining the LSK phenotype in about 85% of totallive cells vs. about 50% for Pten single mutant cultures.

FIG. 2E is a FACS analysis of a 7 week culture of Pten:Ctnnb1 LSK cells(pre-gated on live, lineage negative cells). The boxed area indicatesKit⁺ Sca-1⁺ (LSK) cells.

FIGS. 2F and G are FACS analyses showing a transplant engraftmentexperiment. At 5 weeks culture (see FIG. 2B), Pten and Pten:Ctnnb1 LSKcultures were re-sorted and 1000 LSK cells (CD45.2⁺) from each weretransplanted into lethally irradiated (10Gy) CD45.1⁺ recipient micealong with 2×10⁵ congenic whole bone marrow competitor cells. Becausewild-type cells did not survive 5 weeks culture, 1000 fresh wild-typeLSK cells were also transplanted as a separate control group. At 4 weekspost-transplant, there was no detectable engraftment from peripheralblood analysis of mice transplanted with either Pten or Pten:Ctnnb1 LSKcultures (data not shown). At 5 weeks post-transplant, bone marrow fromrecipient mice was analyzed for donor engraftment (CD45.2⁺ cells) anddonor LSK cells (CD45.2⁺ LSKs). FIGS. 2F and G display representativedonor engraftment (left, boxed areas indicate CD45.2⁺ donor cells) anddonor LSK cell engraftment (right, boxed areas indicate LSK cells) frombone marrow of mice transplanted with 1000 Fresh LSK cells (F) or 1000cultured Pten:Ctnnb1 LSK cells (G).

FIGS. 2H-J are bar graphs showing the quantitative analysis of donor(CD45.2⁺) cells (H), donor LSK cells (I), and fold increase in donorLSKs (J) isolated from bone marrow of recipient mice described in FIGS.2F and 2G at 5 weeks post-transplant.

FIGS. 3A-K are schematics, photographs, bar graphs, and FACS analysesdemonstrating that ex vivo pharmacological manipulation of the PTEN/Aktand Wnt/β-catenin signaling pathways cooperatively drive functional HSCexpansion.

FIG. 3A is a schematic illustrating representative members of the Wntand PTEN pathways. Inhibition of GSK-3β leads to β-catenin activationwhich blocks HSC differentiation. Inhibition of PTEN leads to Aktactivation which promotes survival. Both pathways individually have beenshown to promote HSC proliferation.

FIGS. 3B and C are photographs of HSCs. One hundred LSK Flk2⁻ cells weresorted from wild-type (C57Bl/6) mice and cultured in (1) media, (2)media +1 μM CHIR99021 (GSK-3β inhibitor), (3) media+200 nM DipotassiumBis-peroxo(picolinato)oxovanadate (BpV(pic), a PTEN inhibitor), and (4)media+1 μM CHIR99021+200 nM BpV(pic). An alternative PTEN inhibitor,Shikonin, was also utilized at 200 nM alone (5) or in combination with 1μM CHIR99021 (6). Pictures are at 17 days culture (B, originalmagnification 100×) and 23 days (C, original magnification 40×).Compared to control, both inhibitors applied individually yield greaterexpansion of LSK cells indicating that GSK-3β inhibition is not strictlyequivalent to constitutive activation of β-catenin shown in Ctnnb1mutant LSKs while BpV(pic) yields similar results compared to Ptenmutant LSKs (see FIG. 2). Similar to double mutant LSKs (FIG. 2), thegreatest expansion is shown with both inhibitors present (FIG. 3B/C,panel 4).

FIG. 3D is a series of photographs showing LSK Flk2⁻ cells at 28 daysculture in the indicated media conditions (original magnification 200×).Here, significant expansion relative to control is observed with bothinhibitors present individually; however, significantdifferentiation/heterogeneity of cell morphology is observed in bothsingle inhibitor cultures, including more variable cell size/morphologyand/or differentiation to adherent, spindle-shaped cells (middlepanels). In contrast, and quite surprisingly, expansion with homogeneityis achieved when both inhibitors are present (last panel).

FIG. 3E is a FACS analysis of 28 day LSK Flk2⁻ cells cultured in mediacontaining both inhibitors (200 nM BpV(pic) and 1 μM CHIR99021). Cellswere pre-gated on live, lineage negative cells. The boxed area indicatesKit⁺ Sca1⁺ (LSK) cells. Greater than 90% of LSKs retain Flk2 negativity(data not shown). The LSK Flk2⁻ phenotype is maintained with high purityin cultures containing both inhibitors.

FIG. 3F is a bar graph showing fold expansion of LSK Flk2⁻ cells after28 days culture in the indicated conditions. While both inhibitors addedindividually lead to significant expansion compared to media withouteither inhibitor, the greatest expansion (˜270 fold) is observed whenboth inhibitors are added together.

FIGS. 3G and H are bar graphs showing the % repopulation and % CD45.2⁺cells from engrafted mice. Twenty-eight day cultures (FIGS. 3 D-F) werere-sorted for LSK Flk2⁻ cells and 1000 LSK Flk2⁻ cells (CD45.2⁺) fromeach media condition were transplanted into lethally irradiated (10Gy)CD45.1⁺ recipient mice along with 2×10⁵ congenic whole bone marrowcompetitor cells. At 4 weeks post-transplant, peripheral blood wasanalyzed for donor (G) and multi-lineage (H) engraftment. In FIG. 3G,each bar represents an individual mouse; the horizontal-dashed linerepresents the average “engraftment” of mice transplanted withcompetitor cells only and thus the limit of detectability for trueengraftment. Long-term (4 month) engraftment has not been observed from28-day cultures (data not shown). 6 of 8 mice show >1% engraftment whentransplanted with LSK Flk2⁻ cells cultured with both inhibitors presentcompared to 4/8 with only CHIR99021 present, 0/10 with only BpV(pic)present, and 2/6 with media only. One percent or greater engraftment isa standard limit for substantial engraftment. (Zhang, C. C., et al., NatMed, 12(2): 240-5, 2006. Zhang, C. C. and H. F. Lodish, Blood, 105(11):4314-20, 2005.) Thus, while use of both inhibitors together leads togreatest expansion in LSKs (FIG. 3F), transplantation of equivalentnumbers of these cultured LSK Flk2⁻ cells also yields increasedshort-term engraftment/functionality when cultured with both inhibitorscompared to no or either single inhibitor only.

FIG. 3I is a bar graph showing the fold expansion of LSK Flk2⁻ cellsafter 9 days culture in (1) media, (2) media+200 nM BpV(pic), (3)media+100 nM CHIR99021, and (4) media+200 nM BpV(pic)+100 nM CHIR99021.Because long-term engraftment was not observed from 28 day cultures(FIG. 3D-H and data not shown), LSK Flk2⁻ cells were cultured for only 9days to test if both expansion and long-term repopulation can beachieved. Similar trends are observed here to the 28 day cultures(compare to FIG. 3F) although the extent of expansion is substantiallyreduced at only 9 days compared to 28 days culture.

FIG. 3J is a FACS analysis of 9 day LSK Flk2⁻ cells cultured inmedia+200 nM BpV(pic)+100 nM CHIR99021. The boxed area indicates Kit⁺Sca-1⁺ (LSK) cells. Cells were pre-gated on live, lineage negativecells. Greater than 90% of LSKs retain Flk2 negativity (data not shown).Here, the levels of Sca-1 and Kit appear normal compared to theSca-1^((high))Kit^((high)) population shown from 28 day cultures (FIG.3E).

FIG. 3K is a bar graph showing % repopulation of 10-day cultured cellsin mice. Ten day cultures were transplanted into lethally irradiated(10Gy) CD45.1⁺ recipient mice along with 2×10⁵ congenic whole bonemarrow competitor cells. The total, non-adherent cell product after 10days culture of 100 initial LSK Flk2⁻ cells was transplanted per mouse.At 8 weeks post-transplant, peripheral blood was analyzed for donorengraftment. As in FIG. 3H, multi-lineage reconstitution was observedfrom all mice exhibiting true engraftment (data not shown). Each barrepresents an individual mouse; the horizontal-dashed line representsthe average ‘engraftment’ of mice transplanted with competitor cellsonly and thus the limit of detectability for true engraftment. Here, 3/7mice transplanted with LSK Flk2⁻ cells cultured in the presence of bothinhibitors exhibited 1% or greater donor engraftment vs. no micereaching this threshold in the single or no inhibitor groups.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the invention is a method for expanding a populationof stem cells obtained from a tissue selected from the group consistingof peripheral blood, cord blood, and bone marrow. This method comprisesmodulating a PTEN pathway and a Wnt pathway in the population of stemcells to expand the number of stem cells.

As used herein, “expand”, “expanding” and like terms means to increasethe number of stem cells in the population relative to the number ofstem cells in the original population either in vivo or ex vivo usingany of the methods disclosed herein. Preferably, the expansion is atleast 40-fold compared to the original number of stem cells in thepopulation. More preferably, the expansion is at least 80-fold,100-fold, 150-fold, 200-fold, 250-fold, or 270-fold compared to theoriginal number of stem cells.

In the present invention “a population of stem cells” means a group ofsubstantially undifferentiated cells that possess the ability to giverise to many different types of cells and which have the ability toself-renew. Representative, non-limiting examples of stem cellsaccording to the present invention include bronchioalveolar stem cells(BASCs), bulge epithelial stem cells (bESCs), corneal epithelial stemcells (CESCs), cardiac stem cells (CSCs), epidermal neural crest stemcells (eNCSCs), embryonic stem cells (ESCs), endothelial progenitorcells (EPCs), hepatic oval cells (HOCs), hematopoetic stem cells (HSGs),keratinocyte stem cells (KSCs), mesenchymal stem cells (MSCs), neuronalstem cells (NSCs), pancreatic stem cells (PSCs), retinal stem cells(RSCS), and skin-derived precursors (SKPs).

Hematopoietic stem cells, for example, have the ability to self-renew(i.e., expand) and can give rise to all the types of progenitor cells(such as, e.g., CMP, GMP, MEP and CLP) and ultimately all the types ofblood cells (such as e.g., red blood cells, B lymphocytes, Tlymphocytes, natural killer cells, neutrophils, basophils, eosinophils,monocytes, macrophages, and platelets) in the hematopoietic system.

In the present invention, “modulating”, “modulation” and like terms meanaltering the signal transduction pathway, e.g., a protein in the PTENand/or Wnt pathways, including but not limited to lowering or increasingthe expression level of a protein, altering the sequence of such aprotein (by mutation, pre-translational or post-translationalmodification or otherwise), or inhibiting or activating such a protein(whether by binding, phosphorylation, glycosylation, translocation orotherwise). Such modulation may be achieved genetically orpharmacologically.

As used herein, “a modulator of a PTEN pathway” (or “PTEN pathwaymodulator”) is any agent that regulates the activity of any member ofthe PTEN pathway, which results in, e.g., increased β-catenin expressionin a stem cell, and/or increased β-catenin function in a stem cell,and/or increased β-catenin localization to a nucleus of a stem celland/or provides a survival signal complementary to β-catenin. Thus, amodulator of the PTEN pathway may act upstream or downstream of PTEN;preferably the modulator acts at or downstream from PTEN. Inhibition ofPTEN leads to Akt activation which promotes survival (FIG. 3A).Representative, non-limiting examples of members of the PTEN pathway,include PTEN, phosphatidylinositol 3-kinase (PI3K), the serine/threonineprotein kinase Akt, and 6-catenin.

Representative non-limiting examples of PI3K modulators, particularlyPI3K activators, include pervanadate (Maude Tessier and James R.Woodgett, J. Biol. Chem., 281(33):23978-23989 (2006)), insulin (Hui, L.,et al., Brain Research, 1052(1):1-9 (2005)), insulin-like growth factor(Kenney, A. M., et al., Development, 131:217-228 (2004) and Datta, S.R., et al., Cell, 91:231-241 (1997)), platelet derived growth factor(Datta, S. R., et al., Cell 91:231-241 (1997)), carbachol (Cui, Q L, etal., Neurochem Int, 48:383-393 (2006)), nicotine (West, K. et al., J.Clinical Investigation, 111:81-90 (2003)),4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (Id.),adrenomedullin (AM) (Nikitenko, L L et al., British J. Cancer, 94:1-7(2006)), lysophosphatidic acid, platelet activating factor, macrophagesimulating factor, and sphingosine-1-phosphate.

Representative non-limiting examples of Akt modulators, particularly Aktactivators, include Ro-31-8220 (Wen, H. et al., Cellular signaling,15:37-45 (2003)); Nicotine (West, K. et al., J. Clinical Investigation,111:81-90 (2003)); carbachol (Cui Q L, Fogle E & Almazan G NeurochemInt, 48:383-393 (2006)); 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) (West, K. et al., J. Clinical Investigation, 111:81-90 (2003));adrenomedullin (AM) (Nikitenko, L L et al, British J. Cancer, 94:1-7(2006)); lysophosphatidic acid; platelet activating factor, macrophagesimulating factor; sphingosine-1-phosphate; cAMP-elevating agents, suchas forskolin, chlorophenylthio-cAMP, prostaglandin-El, and 8-bromo-cAMP(Song et al., J. Cell. Mol. Med., 9(1):59-71 (2005)); and growthfactors, including insulin and insulin growth factor-1 (Datta, S. R., etal., Cell, 91:231-241 (1997)), and platelet derived growth factor.

Additional preferred modulators of the present invention include thosethat target mTOR, RHEB, FoxO, p27, BAD, caspase-9, or p53.Representative non-limiting examples of such modulators include mTORmodulators, particularly mTOR activators, such as phosphatidic acid (PA)(see, e.g., WO/2006/027545; Foster, D. A., Cancer Res, 67(1):1-4 (2007);and Tee et al., J. Biol. Chem. 278:37288-96 (2003)); RHEB modulators,particularly RHEB-GTPase inhibitors, such as RHEB antibodies (see, e.g.,WO/2004/048536); FoxO modulators, particularly FoxO inhibitors, such asFKH(DBD), which is a truncated version of FKHRL1 (see, e.g., Gilley, J.,et al., J. Cell Biol. 162(4):613-622 (2003)); p27 modulators,particularly p27 inhibitors, such as p27 antisense inhibitors andtriplex forming oligonucleotides, protein and peptide antagonists (see,e.g., U.S. Pat. No. 5,958,769); BAD modulators, particularly BADinhibitors, such as 14-3-3 protein (see, e.g., S. Hsu et al., MolecularEndocrinology 11 (12):1858-1867 (1997)); caspase-9 modulators,particularly caspase-9 inhibitors, such as LB-84451 (LG Life Sciences)and Z-LEHD-FMK Caspase Inhibitor (Thornberry, N.A., and Lazebnik, Y.,Science 281:1312-1316 (1998)); and p53 modulators, particularly p53inhibitors, such as Pifithrin-α and its derivatives (see, e.g., Science,Komarov et al., 285 (5434): 1733-1737 (1999), Pietrancosta et al., DrugDev Res 65:43-49 (2005)).

In the present invention, “a modulator of a Wnt Pathway” (or “Wntpathway modulator”) is any agent that regulates the activity of anymember of the Wnt pathway, which results in, e.g., increased β-cateninexpression in a stem cell, and/or increased β-catenin function in a stemcell, and/or increased β-catenin localization to a nucleus of a stemcell. A modulator of the Wnt pathway may act upstream or downstream ofWnt. Preferably, the modulator acts at or downstream from GSK-3β.Representative, non-limiting examples of members of a Wnt pathway,include Wnt, seven-transmembrane Frizzled (Fz), the single-pass, LDLreceptor-related proteins (LRP) 5/6, Axin, Dishevelled, glycogensynthase kinase 3 beta (GSK-3β), adenomatous polyposis coli (APC), andβ-catenin. Inhibition of GSK-3β leads to Akt activation which promotessurvival (FIG. 3A).

In one aspect of the present invention, modulating the PTEN pathwaycomprises introducing a mutation into a population of stem cells, whichmutation results in modulation of a molecule in the PTEN pathway. In thepresent invention, modulation of the PTEN pathway also includescontacting the stem cells with a modulator of a molecule in the PTENpathway that leads to β-catenin activation. Representative, non-limitingexamples of such modulators include a small molecule, a biologic, anantisense RNA, a small interfering RNA (siRNA), and combinationsthereof. This aspect of the invention further includes modulating theWnt pathway, which comprises introducing a mutation into a population ofstem cells that results in modulation of a molecule in the Wnt pathway.In the present invention modulation of the Wnt pathway also includescontacting the stem cells with a modulator of a molecule in the Wntpathway. Representative, non-limiting examples of such a modulatorinclude a small molecule, a biologic, an antisense RNA, a smallinterfering RNA (siRNA), and combinations thereof.

As used herein, “introducing a mutation” means any conventional methodfor producing an alteration in the genetic makeup of the stem cellpopulation. Non-limiting examples for introducing a mutation into a stemcell population include mutagenesis via ultra-violet light irradiation,chemical mutagenesis, targeted mutagenesis such as site directedmutagenesis of a stem cell, and creation of a transgenic mouse.

In the present invention, the phrase “modulation of a molecule in thePTEN pathway” means altering the function of a member of the PTENpathway, which altered function has an effect similar to inhibiting ordecreasing the function of PTEN. Non-limiting examples of such“modulation” include constitutively activating β-catenin, constitutivelyactivating Akt, or loss-of-function or null alleles of PTEN. The phrase“modulation of a molecule in the Wnt pathway” means blocking ordecreasing the function of a member of the Wnt pathway, which has aneffect similar to blocking or decreasing GSK-313 function. Non-limitingexamples of such modulation include constitutively activating β-cateninand loss-of-function or null alleles of GSK-3β.

“Modulators of a molecule in the PTEN pathway” are molecules that cause,directly or indirectly, activation of β-catenin. Non-limiting examplesof such molecules include those that activate β-catenin, activate Akt,activate PI3K, or inhibit PTEN. “Modulators of a molecule in the Wntpathway” are molecules that directly or indirectly block or decrease thefunction of a member of the Wnt pathway. Non-limiting examples of suchmolecules include those that activate β-catenin or that inhibit GSK-3β,Axin, or APC.

In the present invention, the term “small molecule” includes anychemical or other moiety, other than polypeptides and nucleic acids,that can act to affect biological processes, particularly to modulatemembers of the Wnt and PTEN pathways. Small molecules can include anynumber of therapeutic agents presently known and used, or that can besynthesized in a library of such molecules for the purpose of screeningfor biological function(s). Small molecules are distinguished frommacromolecules by size. The small molecules of the present inventionusually have a molecular weight less than about 5,000 daltons (Da),preferably less than about 2,500 Da, more preferably less than 1,000 Da,most preferably less than about 500 Da.

Small molecules include without limitation organic compounds,peptidomimetics and conjugates thereof. As used herein, the term“organic compound” refers to any carbon-based compound other thanmacromolecules such as nucleic acids and polypeptides. In addition tocarbon, organic compounds may contain calcium, chlorine, fluorine,copper, hydrogen, iron, potassium, nitrogen, oxygen, sulfur and otherelements. An organic compound may be in an aromatic or aliphatic form.Non-limiting examples of organic compounds include acetones, alcohols,anilines, carbohydrates, monosaccharides, oligosaccharides,polysaccharides, amino acids, nucleosides, nucleotides, lipids,retinoids, steroids, proteoglycans, ketones, aldehydes, saturated,unsaturated and polyunsaturated fats, oils and waxes, alkenes, esters,ethers, thiols, sulfides, cyclic compounds, heterocyclic compounds,imidizoles, and phenols. An organic compound as used herein alsoincludes nitrated organic compounds and halogenated (e.g., chlorinated)organic compounds.

Preferred small molecules are relatively easier and less expensivelymanufactured, formulated or otherwise prepared. Preferred smallmolecules are stable under a variety of storage conditions. Preferredsmall molecules may be placed in tight association with macromoleculesto form molecules that are biologically active and that have improvedpharmaceutical properties. Improved pharmaceutical properties includechanges in circulation time, distribution, metabolism, modification,excretion, secretion, elimination, and stability that are favorable tothe desired biological activity. Improved pharmaceutical propertiesinclude changes in the toxicological and efficacy characteristics of thechemical entity.

In general, a polypeptide mimetic (“peptidomimetic”) is a molecule thatmimics the biological activity of a polypeptide, but that is notpeptidic in chemical nature. While, in certain embodiments, apeptidomimetic is a molecule that contains no peptide bonds (that is,amide bonds between amino acids), the term peptidomimetic may includemolecules that are not completely peptidic in character, such aspseudo-peptides, semi-peptides, and peptoids.

As used herein, the term “biologic” means products derived from livingsources as opposed to a chemical process. Non-limiting examples of a“biologic” include proteins, conditioned media, and partially purifiedproducts from tissues.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein. In the present invention, these terms mean alinked sequence of amino acids, which may be natural, synthetic, or amodification or combination of natural and synthetic. The term includesantibodies, antibody mimetics, domain antibodies, lipocalins, andtargeted proteases. The term also includes vaccines containing a peptideor peptide fragment intended to raise antibodies against the peptide orpeptide fragment.

“Antibody” as used herein includes an antibody of classes IgG, IgM, IgA,IgD, or IgE, or fragments or derivatives thereof, including Fab,F(ab′)2, Fd, and single chain antibodies, diabodies, bispecificantibodies, and bifunctional antibodies. The antibody may be amonoclonal antibody, polyclonal antibody, affinity purified antibody, ormixtures thereof, which exhibits sufficient binding specificity to adesired epitope or a sequence derived therefrom. The antibody may alsobe a chimeric antibody. The antibody may be derivatized by theattachment of one or more chemical, peptide, or polypeptide moietiesknown in the art. The antibody may be conjugated with a chemical moiety.The antibody may be a human or humanized antibody. These and otherantibodies are disclosed in U.S. Published Patent Application No.20070065447.

Other antibody-like molecules are also within the scope of the presentinvention. Such antibody-like molecules include, e.g., receptor traps(such as entanercept), antibody mimetics (such as adnectins, fibronectinbased “addressable” therapeutic binding molecules from, e.g., CompoundTherapeutics, Inc.), domain antibodies (the smallest functional fragmentof a naturally occurring single-domain antibody (such as, e.g.,nanobodies; see, e.g., Cortez-Retamozo et al., Cancer Res. 2004 Apr. 15;64(8):2853-7)).

Suitable antibody mimetics generally can be used as surrogates for theantibodies and antibody fragments described herein. Such antibodymimetics may be associated with advantageous properties (e.g., they maybe water soluble, resistant to proteolysis, and/or be nonimmunogenic).For example, peptides comprising a synthetic beta-loop structure thatmimics the second complementarity-determining region (CDR) of monoclonalantibodies have been proposed and generated. See, e.g., Saragovi et al.,Science. Aug. 16, 1991; 253(5021):792-5. Peptide antibody mimetics alsohave been generated by use of peptide mapping to determine “active”antigen recognition residues, molecular modeling, and a moleculardynamics trajectory analysis, so as to design a peptide mimic containingantigen contact residues from multiple CDRs. See, e.g., Cassett et al.,Biochem Biophys Res Commun. Jul. 18, 2003; 307(1):198-205. Additionaldiscussion of related principles, methods, etc., that may be applicablein the context of this invention are provided in, e.g., Fassina,Immunomethods. October 1994; 5(2):121-9.

As used herein, “peptide” includes targeted proteases, which are capableof, e.g., substrate-targeted inhibition of post-translationalmodification such as disclosed in, e.g., U.S. Patent ApplicationPublication No. 20060275823.

“Antisense” molecules as used herein include antisense or senseoligonucleotides comprising a single-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target mRNA (sense) or DNA(antisense) sequences. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, for example, Stein and Cohen, Cancer Res. 48:2659, (1988)and van der Krol et al., BioTechniques 6:958, (1988).

Antisense molecules can be modified or unmodified RNA, DNA, or mixedpolymer oligonucleotides. These molecules function by specificallybinding to matching sequences resulting in inhibition of peptidesynthesis (Wu-Pong, November 1994, BioPharm, 20-33) either by stericblocking or by activating an RNase H enzyme. Antisense molecules canalso alter protein synthesis by interfering with RNA processing ortransport from the nucleus into the cytoplasm (Mukhopadhyay & Roth,1996, Crit. Rev. in Oncogenesis 7, 151-190). In addition, binding ofsingle stranded DNA to RNA can result in nuclease-mediated degradationof the heteroduplex (Wu-Pong, supra). Backbone modified DNA chemistry,which have thus far been shown to act as substrates for RNase H arephosphorothioates, phosphorodithioates, borontrifluoridates, and2′-arabino and 2′-fluoro arabino-containing oligonucleotides.

Antisense molecules may be introduced into a cell containing the targetnucleotide sequence by formation of a conjugate with a ligand bindingmolecule, as described, e.g., in WO 91/04753. Suitable ligand bindingmolecules include, but are not limited to, cell surface receptors,growth factors, other cytokines, or other ligands that bind to cellsurface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell. Alternatively, a sense or an antisenseoligonucleotide may be introduced into a cell containing the targetnucleic acid sequence by formation of an oligonucleotide-lipid complex,as described, e.g., in WO 90/10448.

The term small interfering RNA (“siRNA”) refers to small inhibitory RNAduplexes that induce the RNA interference (RNAi) pathway. (Elbashir, S.M. et al. Nature 411:494-498 (2001); Caplen, N. J. et al. Proc. Natl.Acad. Sci. USA 98:9742-9747 (2001); Harborth, J. et al. J Cell Sci.114:4557-4565 (2001).) These molecules can vary in length (generally18-30 base pairs) and contain varying degrees of complementarity totheir target mRNA in the antisense strand. Some, but not all, siRNA haveunpaired overhanging bases on the 5′ or 3′ end of the sense strandand/or the antisense strand. The term “siRNA” includes duplexes of twoseparate strands, as well as single strands that can form hairpinstructures comprising a duplex region. As used herein, siRNA moleculesare not limited to RNA molecules but further encompass chemicallymodified nucleotides and non-nucleotides. siRNA gene-targeting may becarried out by transient siRNA transfer into cells (achieved by suchclassic methods as liposome-mediated transfection, electroporation, ormicroinjection).

In another aspect of the present invention, modulating the PTEN and Wntpathways comprises contacting the stem cell population with a smallmolecule inhibitor of the PTEN pathway and a small molecule inhibitor ofthe Wnt pathway. Preferably, modulating the PTEN and Wnt pathwayscomprises down-regulating PTEN and GSK-3β, respectively. As used herein,“down-regulating” means inhibiting or reducing the amount of orinhibiting or decreasing the activity of PTEN and GSK-3β. Suchdown-regulation may be accomplished using, e.g. antisense RNA, siRNA,antibodies, or small molecules.

Preferably, down-regulating PTEN and GSK-3β comprises contacting thestem cell population with: (a) a reversible PTEN inhibitor selected fromthe group consisting of a small molecule, a biologic, an antisense RNA,a small interfering RNA (siRNA), and combinations thereof and (b) areversible GSK-3β inhibitor selected from the group consisting of asmall molecule, a biologic, an antisense RNA, a small interfering RNA(siRNA), and combinations thereof. As used herein, “reversible” meansthat the effect of the down-regulation is not permanent. In the presentinvention, genetic alteration of both the PTEN and the Wnt pathwaysleads to an increased ability to self-renew both in vitro as well as invivo following long-term culture but a failure to differentiate and thusa failure to repopulate the hematopoietic system of transplantrecipients. In contrast, use of reversible down-regulators of bothpathways, such as, e.g., bpV(pic) and CHIR99201, allows for expansion offunctional HSCs, but (1) once the down-regulator is withdrawn, culturedHSCs can differentiate unlike cultured HSCs from genetic mutants, and(2) if such cultured HSC are transplanted, recipient animals do notdevelop leukemia as genetic mutants do.

Preferably, both the reversible PTEN inhibitor and the reversible GSK-3βinhibitor are small molecules. In one aspect, the reversible PTENinhibitor is any molecule, such as a small molecule, which is capable ofinhibiting PTEN or a down-stream member of the PTEN pathway, whichinhibition leads to β-catenin activation. Preferably, the PTEN inhibitoris selected from the group consisting of shikonin, a bisperoxovanadiumcompound, SF-1751 (Semafore Pharmaceuticals), pharmaceutical saltsthereof, and combinations thereof. In this aspect, the bisperoxovanadiumcompound is selected from the group consisting of bpV(phen)₂, bpV(pic),pharmaceutical salts thereof, and combinations thereof.

In the present invention, the reversible GSK-3β inhibitor is anymolecule that is capable of reversibly inhibiting GSK-3β. Preferably,such an inhibitor is selected from the group consisting ofHymenialdisine, Flavopiridol, Kenpaullone, Alsterpaullone,Azakenpaullone, Indirubin-30-oxime, 6-Bromoindirubin-30-oxime (B10),6-Bromoindirubin-30-acetoxime, Aloisine A, Aloisine B, TDZD8, Compound12, CHIR98014, CHIR99021 (CT99021), CT20026, Compound 1, SU9516,ARA014418, Staurosporine, Compound 5a, Compound 29, Compound 46,GF109203x (bisindolylmaleimide I), Ro318220 (bisindolylmaleimide IX),SB216763, SB415286, 15, CGP60474, Compound 8b, TWS119, Compound 1A,Compound 17, Lithium, Beryllium, Zinc, small molecule GSK-3β inhibitors(Vertex Pharmaceuticals), NP-12 (Neuropharma), GSK-3β inhibitors(Amphora), GSK-3β inhibitors (CrystalGenomics), SAR-502250(Sanofi-Aventis), 3544 (Hoffmann-La Roche), GSK-3β inhibitors(Lundbeck), TDZD-8 (Cancer Center, University of Rochester),pharmaceutically acceptable salts thereof, and combinations thereof.

While the PTEN and GSK-3β inhibitors may be contacted with the stem cellpopulation in any convenient manner that achieves the desired level ofstem cell expansion, it is preferred that the inhibitors areco-administered. Moreover, multiple GSK-3β and PTEN inhibitors may becontacted with the stem cells. Furthermore, the PTEN and GSK-3βinhibitors may be contacted/administered to the stem cells in concertwith other agents suitable for promoting stem cell self renewal.Preferably, the PTEN inhibitor is bpV(pic) and the GSK-3β inhibitor isCHIR99201.

In an additional aspect of the present invention, the number of stemcells is increased by a factor of at least 40-fold. Preferably, thenumber of stem cells is increased by a factor of at least 80-fold, suchas at least 100-fold, including at least 150-fold, at least 200-fold, atleast 250-fold, or at least 270-fold. Surprisingly and unexpectedly suchlevels of stem cell expansion are achieved using the methods of thepresent invention.

As noted above, the methods of the present invention may be used toexpand any population of stem cells. Preferably, the stem cells that maybe expanded according to the methods of the present invention mayselected from hematopoietic stem cells (HSGs), endothelial progenitorcells (EPCs), mesenchymal stem cells (MSCs), cardiac stem cells (CSCs),neuronal stem cells (NSCs), and combinations thereof. More preferably,the stem cells are HSCs.

Another embodiment of the invention is a method for ex vivo expansion ofa substantially undifferentiated stem cell population. This methodcomprises modulating a PTEN pathway and a Wnt pathway in theundifferentiated stem cell population to expand the number ofundifferentiated stem cells without significant differentiation of thestem cell population.

In this embodiment, a stem cell population is “substantiallyundifferentiated” if a sufficient number of cells in that populationretain the ability to self-renew and can give rise to variousdifferentiated cell types when transplanted into a recipient, forexample, in the case of an HSC population, repopulating the HSC lineagewhen transplanted. As used herein, “without significant differentiation”means the expanded stem cell population has a sufficient number of cellsthat maintain a multi-lineage differentiation potential that the fullscope of a target stem lineage may be regenerated upon transplantationof the expanded stem cell population into a recipient. Thus, e.g., inthe case of an HSC population, the expanded HSC population, whentransplanted into a recipient, is capable of regenerating the entirehematopoietic cell lineage.

A further embodiment of the invention is a method for ex vivo expansionof an hematopoietic stem cell (HSC) population obtained from a tissueselected from the group consisting of peripheral blood, cord blood, andbone marrow. This method comprises modulating a PTEN pathway and a Wntpathway in the HSC population to expand the HSC population to asufficient quantity while maintaining a multilineage differentiationpotential in the HSC population, which is sufficient for subsequenttransplantation into a patient in need thereof.

As used herein, “obtained” from a tissue means any conventional methodof harvesting or partitioning tissue from a donor. For example, thetissue may obtained from a blood sample, such as a peripheral or cordblood sample, or harvested from bone marrow. Methods for obtaining suchsamples are well known to the artisan. In the present invention, thesamples may be fresh, i.e., obtained from the donor without freezing.Moreover, the samples may be further manipulated to remove extraneous orunwanted components prior to expansion. The samples may also be obtainedfrom a preserved stock. For example, in the case of peripheral or cordblood, the samples may be withdrawn from a cryogenically or otherwisepreserved bank of such blood. Such samples may be obtained from anysuitable donor. Preferably, the donor is a mammal, for example, aprimate, such as a human. Furthermore, the sample may be obtained froman autologous or allogeneic donor or source. Preferably, the sample isobtained from an autologous source.

In this method, “maintaining a multilineage differentiation potential”means that the expanded HSC population has the ability, whentransplanted into a patient in need of such a transplant, to regenerateall the types of progenitor cells e.g., CMP, GMP, MEP, and CLP, andultimately all the types of blood cells including, e.g., red bloodcells, B lymphocytes, T lymphocytes, natural killer cells, neutrophils,basophils, eosinophils, monocytes, macrophages, and platelets in thehematopoietic system.

In the present invention, that quantity of expanded HSCs, which is“sufficient for subsequent transplantation” generally corresponds tothat number of HSCs, which would result in greater than about 1%engraftment after transplantation. This is one accepted measure of asuccessful transplant. In the present invention, any conventional methodmay be used to determine the % engraftment, including the one set forthin the Examples. Such a measure may be carried out with or withoutcompetitor cells, typically and preferably, without competitor cells.(Zhang, C. C., et al., Nat Med, 12(2): 240-5, 2006. Zhang, C. C. and H.F. Lodish, Blood, 105(11): 4314-20, 2005).

In the above described ex vivo expansion methods, modulating the PTENand Wnt pathways may be achieved as previously set forth. Modulating thePTEN and Wnt pathways may include contacting the stem cell populationwith a small molecule inhibitor of the PTEN pathway and a small moleculeinhibitor of the Wnt pathway. Modulating the PTEN and Wnt pathways mayinclude down-regulating PTEN and GSK-38, respectively. Preferably,down-regulating the PTEN and Wnt pathways comprises contacting the stemcell population with a reversible PTEN inhibitor and a reversible GSK-3βinhibitor as previously described. Preferably, both the reversible PTENinhibitor and the reversible GSK-3β inhibitor are small molecules.

The reversible PTEN inhibitor may be selected from the group consistingof shikonin, a bisperoxovanadium compound, SF-1751 (SemaforePharmaceuticals), pharmaceutical salts thereof, and combinationsthereof. Preferably, the bisperoxovanadium compound is selected from thegroup consisting of bpV(phen)2, bpV(pic), pharmaceutical salts thereof,and combinations thereof.

The reversible GSK-3β inhibitor may be selected from the groupconsisting of Hymenialdisine, Flavopiridol, Kenpaullone, Alsterpaullone,Azakenpaullone, Indirubin-30-oxime, 6-Bromoindirubin-30-oxime (B10),6-Bromoindirubin-30-acetoxime, Aloisine A, Aloisine B, TDZD8, Compound12, CHIR98014, CHIR99021 (CT99021), CT20026, Compound 1, SU9516,ARA014418, Staurosporine, Compound 5a, Compound 29, Compound 46,GF109203x (bisindolylmaleimide I), Ro318220 (bisindolylmaleimide IX),SB216763, SB415286, I5, CGP60474, Compound 8b, TWS119, Compound 1A,Compound 17, Lithium, Beryllium, Zinc, small molecule GSK-3β inhibitors(Vertex Pharmaceuticals), NP-12 (Neuropharma), GSK-3β inhibitors(Amphora), GSK-3β inhibitors (CrystalGenomics), SAR-502250(Sanofi-Aventis), 3544 (Hoffmann-La Roche), GSK-3β inhibitors(Lundbeck), TDZD-8 (Cancer Center, University of Rochester),pharmaceutically acceptable salts thereof, and combinations thereof.

In these ex vivo expansion methods, preferably, the PTEN inhibitor isbpV(pic), and the GSK-3β inhibitor is CHIR99201. In these methods, it ispreferred that the stem cell is selected from HSCs, endothelialprogenitor cells, (EPCs), mesenchymal stem cells (MSCs), cardiac stemcells (CSCs), neuronal stem cells (NSCs), and combinations thereof.Preferably the stem cell is an HSC. In these methods, the HSC isobtained from a mammalian, e.g., primate or human, tissue selected fromthe group consisting of cord blood, peripheral blood, and bone marrow.

In another aspect of the method for ex vivo expansion of anhematopoietic stem cell (HSC) population, the expansion of the number ofstem cells is by at least 40-fold, such as e.g., by at least 80-fold,including at least 100-fold, at least 150-fold, at least 200-fold, atleast 250-fold, or at least 270-fold.

Yet another embodiment of the present invention is an expanded,substantially undifferentiated stem cell population made by a method ofthe present invention, such as, e.g., the method for ex vivo expansionof a substantially undifferentiated stem cell population or the methodfor ex vivo expansion of an hematopoietic stem cell (HSC) population.

An additional embodiment of the present invention is a method for exvivo expansion of hematopoietic stem cells (HSCs) by at least 40-fold,wherein the expanded HSCs, are competent to reconstitute an HSC lineageupon transplantation into a mammalian patient in need thereof. Thismethod comprises culturing a population of HSCs in a suitable culturemedium comprising a PTEN inhibitor and a GSK-3β inhibitor.

In this aspect of the invention, “competent to reconstitute an HSClineage” means that the expanded HSCs, when transplanted into a suitablemammalian patient, result in greater than 1% engraftment in therecipient, which engrafted cells are able to differentiate into the celllineages necessary to have a normal functioning hematopoietic system. Inthis method, a “suitable culture medium”, “fluid media” and “media”which are used interchangeably herein, mean physiologically balancedsalt solutions that can maintain a stem cell population for a requiredperiod of time, which solution may be supplemented with the PTEN andGSK-3β modulator/inhibitors of the present invention. Such base culturemedia are well known in the arts. A non-limiting example of a suitablebase culture medium for HSCs is StemSpan Media (Stem Cell Technologies;Cat. No. 09600), which is supplemented with 10 ug/ml Heparin, 5×Penicillin/Streptomycin, 10 ng/ml recombinant mouse (rm) Stem CellFactor, and 20 ng/ml rm-Thrombopoietin.

Typically, the culture media also includes from about 100 to about 1000nM of the PTEN inhibitor. The culture media may further include fromabout 50 nM to about 500 nM of the GSK-3β inhibitor. In the presentinvention, when a range is recited, any value within that range,including the endpoints, is contemplated. Preferably, the culture mediaincludes both the PTEN and the GSK-3β inhibitors at the concentrationsindicated. For example, the media may contain as the PTEN inhibitor,bpV(pic), and as the GSK-3β inhibitor, CHIR99201.

In one aspect of this embodiment, the HSCs are obtained from a mammaliantissue, preferably primate or human tissue, which is selected from cordblood, peripheral blood, and bone marrow. In this embodiment, the numberof HSCs is expanded by a factor of at least 80-fold, such as at least100-fold, including at least 150-fold, at least 200-fold, at least250-fold, or at least 270-fold.

Yet another embodiment of the present invention is a kit for expandingan hematopoietic stem cell (HSC) population for subsequenttransplantation into a patient in need thereof. The kit comprises a PTENinhibitor and a GSK-3β inhibitor as described above and instructions forthe use of the inhibitors. Preferably, in the kit, the PTEN inhibitor isbpV(pic) and the GSK-3β inhibitor is CHIR99201. The kit and thecomponents therein may be packaged in any suitable manner fordistribution and/or storage.

A further embodiment of the present invention is a media for carryingout ex vivo expansion of a stem cell population. The media comprises afluid media suitable for maintaining viable stem cells and PTEN andGSK-3β inhibitors present in the media at concentrations sufficient toenable expansion of the stem cell population while maintaining amultilineage differentiation potential in the stem cells.

In this embodiment, a “concentration sufficient to enable expansion”means the minimum concentration of the PTEN and GSK-3β inhibitors, whichare sufficient to achieve the desired level of stem cell renewal, e.g.,expansion sufficient for successful engraftment.

In one aspect of this embodiment, expansion of the number of stem cellsis by a factor selected from the group consisting of at least 40-fold,at least 80-fold, at least 100-fold, at least 150-fold, at least200-fold, at least 250-fold, or at least 270-fold.

A further embodiment of the present invention is a method foradministering an hematopoietic stem cell (HSC) to a patient in needthereof. The method comprises (a) culturing, in a suitable culturemedia, a sample containing an HSC population in the presence of amodulator of a molecule in the PTEN pathway and a modulator of amolecule in the Wnt pathway for a period of time sufficient to expandthe number of HSCs in the sample to a number sufficient to transplantinto the patient; (b) removing from the culture the PTEN and Wnt pathwaymodulators; and (c) administering the HSCs to the patient. In thisembodiment, the culture media, sample, and PTEN and GSK-3β modulatorsare previously described.

An additional embodiment of the present invention is a method forreconstituting bone marrow in a patient in need thereof. The methodcomprises culturing, in a suitable culture media, a sample containing anHSC population in the presence of a modulator of a molecule in the PTENpathway and a modulator of a molecule in the Wnt pathway for a period oftime sufficient to expand the number of HSCs in the sample to a numberthat is sufficient to transplant into the patient. Next, the PTEN andWnt pathway modulators are removed from the culture. Then, the expandedHSCs are administered to the patient in any conventional manner.

In this method, “reconstituting bone marrow” means restoration of all ora portion of the bone marrow in a patient suffering from a disease inwhich normal bone marrow function has been compromised. Non-limitingexamples of such diseases include aplastic anemia, myelodysplasticsyndromes (MDS), paroxysmal nocturnal hemoglobinuria (PNH), and bloodcancers, such as leukemia. Thus, as used herein, “reconstituted” meansthat the transplanted HSCs are able to successfully engraft in the hostand differentiate into all the cell lineages typically found in orderived from bone marrow.

In this method, “a period of time sufficient to expand the number ofHSCs” means the minimum amount of time to expand the HSCs in culture toa point where there is a sufficient number of HSCs for one or moretransplantations. Typically, such a period of time may be at least about10 days in culture. Under certain circumstances, it may be desirable toexpand the stem cell, e.g., HSC, population beyond what is required fora single transplantation. For example, it may be desirable to expand thestem cell, e.g., HSC, population to a number sufficient for multipletransplantations, such as e.g., from about 2 to about 100transplantations. In these circumstances, the excess cells may bepreserved for later use by any conventional method, such as e.g., bycryo-preservation.

As indicated previously, “a number sufficient to transplant” means theminimum number of stem cells, e.g., HSCs, necessary to achieve greaterthan 1% engraftment in a recipient. “Administering the HSCs to thepatient” means conventional methods for delivering HSCs to the patient,including but not limited to, delivering the HSCs surgically and/orintravenously. In this embodiment, the tissue the HSCs are obtainedfrom, and the PTEN and GSK-3β inhibitors are as previously described.

An additional embodiment of the present invention is a method forexpanding a population of hematopoietic stem cells (HSCs). This methodcomprises culturing a population of HSCs under conditions sufficient toresult in an expansion of the HSC population by at least 40-fold,wherein the expanded population of HSCs is suitable for transplantationinto a mammal in need thereof. In this embodiment the “conditionssufficient to result in an expansion of the HSC population” are thoseconditions that can result in expansion of HSCs in culture by, e.g., atleast 40-fold, such as, e.g., by at least 80-fold, at least 150-fold, atleast 200-fold, at least 250-fold, or at least 270-fold. “Suitable fortransplantation into a mammal” means that the number and quality of HSCsis sufficient to support greater than 1% engraftment in a mammalianrecipient, such as, e.g., a primate recipient, including an humanrecipient, in need thereof.

Yet another embodiment of the present invention is a method for treatinga patient in need of a bone marrow transplant, a peripheral bloodtransplant, or a cord blood transplant comprising administering to thepatient a population of HSCs obtained by a method disclosed herein,particularly the methods for expanding a population of hematopoieticstem cells (HSCs).

A further embodiment of the present invention is a method for expandinga population of hematopoietic stem cells (HSCs). The method comprises(a) obtaining from a mammal a tissue sample comprising an HSCpopulation; (b) expanding, in vitro, the HSC population from the sample,wherein (i) the HSC population expands by at least 40-fold; and (ii) theexpanded HSC population has the ability to reconstitute an hematopoieticlineage for at least 4-weeks, such as for example at least 8-weeks,after transplantation into a recipient. In this embodiment the “abilityto reconstitute an hematopoietic lineage” means that the expanded HSCpopulation when transplanted into a recipient will result in greaterthan 1% engraftment of HSC in a recipient. In one aspect of thisembodiment, the HSC population expands by at least 80-fold, such ase.g., at least 100-fold, including at least 150-fold, at least 200-fold,at least 250-fold, or at least 270-fold. In another aspect of thisembodiment, the mammal is a primate, including a human. Preferably, thehuman requires a peripheral blood transplant, a cord blood transplant,or a bone marrow transplant. In a further aspect, the tissue sample isobtained from a tissue selected from the group consisting of cord blood,peripheral blood, and bone marrow.

An additional embodiment of the present invention is a method forreconstituting an hematopoietic stem cell lineage in a recipient in needthereof. The method comprises (a) obtaining from a mammal a tissuesample comprising an HSC population; (b) expanding, in vitro, the HSCpopulation from the sample, wherein: (i) the HSC population expands byat least 40-fold, such as for example, by at least 80-fold, including atleast 100-fold, at least 150-fold, at least 200-fold, at least 250-fold,or at least 270-fold, and (ii) the expanded HSC population has theability to reconstitute an hematopoietic lineage for at least 4-weeks,for example, at least 8-weeks, after transplantation into a recipient inneed thereof; and (c) transplanting the expanded HSC population into arecipient such as a mammal, including a primate or human, in needthereof.

In this embodiment, “reconstituting an hematopoietic stem cell lineage”means that the expanded HSCs, when transplanted into a recipient resultin greater than 1% engraftment of hematopoietic cells, which are able todifferentiate into the normal hematopoietic lineages. In thisembodiment, the human recipient requires a peripheral blood transplant,a cord blood transplant or a bone marrow transplant. Thus, in a furtheraspect, the tissue sample is obtained from a tissue selected from thegroup consisting of cord blood, peripheral blood, and bone marrow. Thesample may be obtained from an autologous or allogeneic source.Preferably, the sample is obtained from an autologous source.

In the present invention, it is preferred that the expanded HSCpopulation comprises HSCs that have a phenotype selected from the groupconsisting of CD34⁻ orCD34⁺/CD38^(−/low)/Thy-1⁺/CD90⁺/Kit^(−/lo)/Lin⁻/CD133⁺ VEGFR2⁺, whichare markers for the most primitive and long-term undifferentiated humanHSCs; CD150⁺/CD48⁻/CD244⁻, which is a marker for human HSCs and theirprogenitors; and/or CD150⁻/CD48⁻/CD244⁺ and CD150⁻/CD48⁺/CD244⁺, whichare markers for non-self-renewing multipotent hematopoietic progenitors,and combinations thereof. (See, e.g., Mimeault, M., et al., Stem Cells:A Revolution in Therapeutics—Recent Advances in Stem Cell Biology andTheir Therapeutic Applications in Regenerative Medicine and CancerTherapies. Clin Pharmacol Ther., 82(3):252-64 (2007)).

The exact proportions of HSCs having these markers in the population isnot critical, so long as the expanded HSC population as a whole issufficient to result in at least 1% engraftment in a recipient.

In another embodiment, the invention is a method for expanding ahematopoietic stem cell population in a mammal in need of suchexpansion. This method comprises administering to the mammal atherapeutically effective amount of a modulator of Wnt and Akt for aperiod of time sufficient to expand the HSC population by at least40-fold with HSCs that possess the ability to reconstitute anhematopoietic lineage in the mammal.

In this method, the respective modulators of Wnt and Akt may be anymolecule, such as a small molecule, a biologic, an antisense RNA, asiRNA, or combinations thereof, which acts directly or indirectly toactivate β-catenin. Preferably, the Wnt modulator is selected from a Wntpolypeptide, QS11 (Zhang, Q. et al., PNAS, 104(18):7444-8 (2007)),2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine(Liu, J. et al., Angew Chem Int Ed Engl. 44(13):1987-90 (2005)),deoxycholic acid (R. Pai et al., Mol Biol Cell. 15(5):2156-63 (2004)),and combinations thereof. Preferably, the modulator of Akt is selectedfrom the group consisting of Ro-31-8220 (Wen, H. et al., Cellularsignaling, 15:37-45 (2003)); Nicotine (West, K. et al., J. ClinicalInvestigation, 111:81-90 (2003)); carbachol (Cui Q L, Fogle E & AlmazanG Neurochem Int, 48:383-393 (2006));4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (West, K. et al.,J. Clinical Investigation, 111:81-90 (2003)); adrenomedullin (AM)(Nikitenko, L L et al, British J. Cancer, 94:1-7 (2006));lysophosphatidic acid; platelet activating factor, macrophage simulatingfactor; sphingosine-1-phosphate; cAMP-elevating agents, such asforskolin, chlorophenylthio-cAMP, prostaglandin-El, and 8-bromo-cAMP(Song et al., J. Cell. Mol. Med., 9(1):59-71 (2005)); and growthfactors, including insulin and insulin growth factor-1 (Datta, S. R., etal., Cell, 91:231-241 (1997)), platelet derived growth factor, andcombinations thereof.

In this method, the Wnt and Akt modulators may be administered using anyregimen that effectively expands the HSC population by at least 40-foldwith HSCs that possess the ability to reconstitute an hematopoieticlineage in the mammal. Preferably, the Wnt and Akt modulators areco-administered.

In the present invention, a “therapeutically effective amount” is anamount sufficient to effect beneficial or desired results. In terms oftreatment of a mammal, a “therapeutically effective amount” of amodulator is an amount sufficient to treat, manage, palliate,ameliorate, or stabilize a condition, such as a bone marrow disease, inthe mammal. A therapeutically effective amount can be administered inone or more doses.

The therapeutically effective amount is generally determined by aphysician on a case-by-case basis and is within the skill of one in theart. Several factors are typically taken into account when determiningan appropriate dosage. These factors include age, sex and weight of thepatient, the condition being treated, the severity of the condition andthe form of the drug being administered.

Effective dosage forms, modes of administration, and dosage amounts maybe determined empirically, and making such determinations is within theskill of the art. It is understood by those skilled in the art that thedosage amount will vary with the route of administration, the rate ofexcretion, the duration of the treatment, the identity of any otherdrugs being administered, the age, size, and species of animal, and likefactors well known in the arts of medicine and veterinary medicine. Ingeneral, a suitable dose of a modulator according to the invention willbe that amount of the modulator, which is the lowest dose effective toproduce the desired effect. The effective dose of a modulator maybeadministered as two, three, four, five, six or more sub-doses,administered separately at appropriate intervals throughout the day.

A modulator, particularly a Wnt or Akt modulator of the presentinvention, may be administered in any desired and effective manner: aspharmaceutical compositions for oral ingestion, or for parenteral orother administration in any appropriate manner such as intraperitoneal,subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal,vaginal, sublingual, intramuscular, intravenous, intraarterial,intrathecal, or intralymphatic. Further, a modulator, particularly a Wntor Akt modulator, of the present invention may be administered inconjunction with other treatments. A modulator, particularly a Wnt orAkt modulator, of the present invention maybe encapsulated or otherwiseprotected against gastric or other secretions, if desired.

While it is possible for a modulator, particularly a Wnt or Aktmodulator, of the invention to be administered alone, it is preferableto administer the modulator as a pharmaceutical formulation(composition). Such pharmaceutical formulations typically comprise oneor more modulators as an active ingredient in admixture with one or morepharmaceutically-acceptable carriers and, optionally, one or more othercompounds, drugs, ingredients and/or materials. Regardless of the routeof administration selected, the modulator, particularly a Wnt or Aktmodulator, of the present invention is formulated intopharmaceutically-acceptable dosage forms by conventional methods knownto those of skill in the art. See, e.g., Remington's PharmaceuticalSciences (Mack Publishing Co., Easton, Pa.).

Pharmaceutically acceptable carriers are well known in the art (see,e.g., Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton,Pa.) and The National Formulary (American Pharmaceutical Association,Washington, D.C.)) and include sugars (e.g., lactose, sucrose, mannitol,and sorbitol), starches, cellulose preparations, calcium phosphates(e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogenphosphate), sodium citrate, water, aqueous solutions (e.g., saline,sodium chloride injection, Ringer's injection, dextrose injection,dextrose and sodium chloride injection, lactated Ringer's injection),alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol),polyols (e.g., glycerol, propylene glycol, and polyethylene glycol),organic esters (e.g., ethyl oleate and tryglycerides), biodegradablepolymers (e.g., polylactide-polyglycolide, poly(orthoesters), andpoly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils(e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut),cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones,talc, silicylate, etc. Each pharmaceutically acceptable carrier used ina pharmaceutical composition comprising a modulator of the inventionmust be “acceptable” in the sense of being compatible with the otheringredients of the formulation and not injurious to the subject.Carriers suitable for a selected dosage form and intended route ofadministration are well known in the art, and acceptable carriers for achosen dosage form and method of administration can be determined usingordinary skill in the art.

Pharmaceutical compositions comprising a modulator of the invention may,optionally, contain additional ingredients and/or materials commonlyused in pharmaceutical compositions. These ingredients and materials arewell known in the art and include (1) fillers or extenders, such asstarches, lactose, sucrose, glucose, mannitol, and silicic acid; (2)binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, sodium starch glycolate, cross-linked sodiumcarboxymethyl cellulose and sodium carbonate; (5) solution retardingagents, such as paraffin; (6) absorption accelerators, such asquaternary ammonium compounds; (7) wetting agents, such as cetyl alcoholand glycerol monosterate; (8) absorbents, such as kaolin and bentoniteclay; (9) lubricants, such as talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, and sodium lauryl sulfate; (10)suspending agents, such as ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth;(11) buffering agents; (12) excipients, such as lactose, milk sugars,polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins,cocoa butter, starches, tragacanth, cellulose derivatives, polyethyleneglycol, silicones, bentonites, silicic acid, talc, salicylate, zincoxide, aluminum hydroxide, calcium silicates, and polyamide powder; (13)inert diluents, such as water or other solvents; (14) preservatives;(15) surface-active agents; (16) dispersing agents; (17) control-releaseor absorption-delaying agents, such as hydroxypropylmethyl cellulose,other polymer matrices, biodegradable polymers, liposomes, microspheres,aluminum monosterate, gelatin, and waxes; (18) opacifying agents; (19)adjuvants; (20) wetting agents; (21) emulsifying and suspending agents;(22), solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan; (23) propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane; (24) antioxidants; (25) agents which render theformulation isotonic with the blood of the intended recipient, such assugars and sodium chloride; (26) thickening agents; (27) coatingmaterials, such as lecithin; and (28) sweetening, flavoring, coloring,perfuming and preservative agents. Each such ingredient or material mustbe “acceptable” in the sense of being compatible with the otheringredients of the formulation and not injurious to the subject.Ingredients and materials suitable for a selected dosage form andintended route of administration are well known in the art, andacceptable ingredients and materials for a chosen dosage form and methodof administration may be determined using ordinary skill in the art.

Pharmaceutical compositions suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, powders, granules, asolution or a suspension in an aqueous or non-aqueous liquid, anoil-in-water or water-in-oil liquid emulsion, an elixir or syrup, apastille, a bolus, an electuary or a paste. These formulations may beprepared by methods known in the art, e.g., by means of conventionalpan-coating, mixing, granulation or lyophilization processes.

Solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like) may be prepared by mixing theactive ingredient(s) with one or more pharmaceutically-acceptablecarriers and, optionally, one or more fillers, extenders, binders,humectants, disintegrating agents, solution retarding agents, absorptionaccelerators, wetting agents, absorbents, lubricants, and/or coloringagents. Solid compositions of a similar type maybe employed as fillersin soft and hard-filled gelatin capsules using a suitable excipient. Atablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared using asuitable binder, lubricant, inert diluent, preservative, disintegrant,surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine. The tablets, and other solid dosageforms, such as dragees, capsules, pills and granules, may optionally bescored or prepared with coatings and shells, such as enteric coatingsand other coatings well known in the pharmaceutical-formulating art.They may also be formulated so as to provide slow or controlled releaseof the active ingredient therein. They may be sterilized by, forexample, filtration through a bacteria-retaining filter. Thesecompositions may also optionally contain opacifying agents and may be ofa composition such that they release the active ingredient only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. The active ingredient can also be inmicroencapsulated form.

Liquid dosage forms for oral administration includepharmaceutically-acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. The liquid dosage forms may containsuitable inert diluents commonly used in the art. Besides inertdiluents, the oral compositions may also include adjuvants, such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents. Suspensions maycontain suspending agents.

Pharmaceutical compositions for rectal or vaginal administration may bepresented as a suppository, which maybe prepared by mixing one or moreactive ingredient(s) with one or more suitable nonirritating carrierswhich are solid at room temperature, but liquid at body temperature and,therefore, will melt in the rectum or vaginal cavity and release theactive compound. Pharmaceutical compositions which are suitable forvaginal administration also include pessaries, tampons, creams, gels,pastes, foams or spray formulations containing suchpharmaceutically-acceptable carriers as are known in the art to beappropriate.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, drops and inhalants. The active compound may be mixed understerile conditions with a suitable pharmaceutically-acceptable carrier.The ointments, pastes, creams and gels may contain excipients. Powdersand sprays may contain excipients and propellants.

Pharmaceutical compositions suitable for parenteral administrationscomprise one or more modulator in combination with one or morepharmaceutically-acceptable sterile isotonic aqueous or non-aqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain suitable antioxidants,buffers, solutes which render the formulation isotonic with the blood ofthe intended recipient, or suspending or thickening agents. Properfluidity can be maintained, for example, by the use of coatingmaterials, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants. These compositions mayalso contain suitable adjuvants, such as wetting agents, emulsifyingagents and dispersing agents. It may also be desirable to includeisotonic agents. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption.

In some cases, in order to prolong the effect of a drug containing amodulator of the present invention, it is desirable to slow itsabsorption from subcutaneous or intramuscular injection. This may beaccomplished by the use of a liquid suspension of crystalline oramorphous material having poor water solubility.

The rate of absorption of the drug then depends upon its rate ofdissolution which, in turn, may depend upon crystal size and crystallineform. Alternatively, delayed absorption of a parenterally-administereddrug may be accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms may be made by forming microencapsulematrices of the active ingredient in biodegradable polymers. Dependingon the ratio of the active ingredient to polymer, and the nature of theparticular polymer employed, the rate of active ingredient release canbe controlled. Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissue. The injectable materials can be sterilized forexample, by filtration through a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the type described above.

The following examples are provided to further illustrate the methodsand compositions of the present invention. These examples areillustrative only and are not intended to limit the scope of theinvention in any way.

EXAMPLES Example 1 Loss of PTEN with Constitutively Active β-CateninLeads to HSC Expansion with Loss of Early Hematopoietic ProgenitorsInduction Of PTEN/Constitutively Active β-Catenin Double Mutant Mice

Mice with homozygous floxed (fl) alleles of Pten (Pten^(fl/fl)) werebred with Ctnnb1^(fl/fl) mice in which exon 3 of the mouse β-cateningene (where all phosphorylation target serine/threonine residues arelocated) was sandwiched by two loxP sequences. (Harada, N., et al., EmboJ, 18(21): 5931-42 1999. Yilmaz, O. H., et al., Nature, 441:475-82 2006.Zhang, J., et al., Nature, 441(7092): 518-22 2006.) Double heterozygousmice from this cross were then crossed to generate Pten^(fl/fl)Ctnnb1^(fl/+) mice (since Ctnnb1 is a gain-of-function allele, onlyheterozygous mice for Ctnnb1 are necessary). Concurrently, Pten^(fl/fl)mice were bred with Scl-Cre⁺ transgenic mice to generate Scl-Cre⁺Pten^(fl/+) mice. These were then crossed to generate Scl-Cre+Pten^(fl/fl) mice (“Pten”). Finally, Pten^(fl/fl) Ctnnb1^(f/+) mice werebred with Scl-Cre⁺ Pten^(fl/fl) mice to generate Scl-Cre⁺ Pten^(fl/fl)Ctnnb1^(fl/+) mice (“Pten:Ctnnb1”). Scl-Cre mice were also bred withCtnnb1^(fl/fl) mice to generate the single mutant Scl-Cre⁺ Ctnnb1^(fl/+)mice (“Ctnnb1”). Mice lacking Scl-Cre (“Scl-Cre negative” or “Control”)were used as controls.

Scl-Cre is an HSC-specific Tamoxifen inducible Cre-recombinase used toachieve conditional knockout of LoxP flanked (floxed) Pten and Ctnnb1alleles. (Gothert, J. R., et al., “In vivo fate-tracing studies usingthe Scl stem cell enhancer: embryonic hematopoietic stem cellssignificantly contribute to adult hematopoiesis.” Blood, 2005. 105(7):p. 2724-2732). Pten/constitutively active β-catenin double mutant micewere induced by intra-peritoneal injection of Tamoxifen (Sigma, Cat. No.T5648) everyday for 5 days using 5 mg on day 1 and 2 mg on days 2-5 eachdissolved in 0.1 ml of corn oil (Sigma, Cat. No. C8267) (completedissolution was achieved by 42° C. water bath sonication for about 5minutes).

HSC Analysis in Bone Marrow and Spleen

Six weeks post-induction of Tamoxifen, bone marrow and spleens wereharvested and made into single cell suspensions. Red blood cell lysiswas performed using hemolysis buffer (0.16M ammonium chloride, SigmaCat. No. A9434). Cells were stained for lineage markers using CD3, CD4,CD8, B220, IgM, Mac-1, Gr1, and Ter119 antibodies along with Kit, andSca-1 for LSK analysis or these markers along with IL-7Ra, CD34 andCD16/32 for progenitor analysis (Akashi, K., et al., A clonogenic commonmyeloid progenitor that gives rise to all myeloid lineages. Nature 2000.404(6774): p. 193-7). Flk2 was added as indicated for LT-HSC analysis.

Unless otherwise indicated, all antibodies were obtained fromeBiosciences (San Diego, Calif.) as indicated below: Fluoresceinisothiocyanate (FITC) conjugated CD3 antibody (Cat. No. 11-0452-85),FITC conjugated CD4 antibody (Cat. No. 11-0042-85), FITC conjugated CD8antibody (Cat. No. 11-0081-85), FITC conjugated B220 antibody (Cat. No.11-0452-85), FITC conjugated Ter119 antibody (Cat. No. 11-5921-85), FITCconjugated Mac-1 antibody (Cat. No. 11-0112-85), FITC conjugated Gr1antibody (Cat. No. 11-5931-85), FITC conjugated IgM antibody (Cat. No.11-5790-85), Phycoerythrin (PE) conjugated Sca-1 antibody (Cat. No.12-5981-83), Allophycocyanin (APC) conjugated Kit antibody (Cat. No.17-1171-83), Biotin conjugated CD135 (Flk-2) antibody (Cat. No.13-1351-85), PE-Cy5 conjugated CD127 (IL-7Ra) antibody (Cat. No.15-1271-83), PE-Cy7 conjugated CD16/32 (FcγRII/III) antibody (Cat. No.25-0161-82), Biotin conjugated CD34 antibody (Cat. No. 13-0341-85),Streptavidin conjugated PE-Cy7 antibody (Cat. No. 25-4317-82),Streptavidin conjugated APC-Cy7 antibody (Cat. No. 10-4317-82), APCconjugated Gr1 antibody (Cat. No. 17-5931-82), APC conjugated B220antibody (Cat. No. 17-0452-83), PE conjugated Mac-1 antibody (Cat. No.12-0112-83), and PE conjugated CD3 antibody (Cat. No. 12-0031-85).

Antibody stained cells were sorted by FACS using a MoFlo (Dako, Ft.Collins, Colo.) flow cytometer and/or a CyAn ADP (Dako, Ft. Collins,Colo.), and analyzed for lineage negative, Sca-1⁺ Kit⁺ (LSK) cells inScl-Cre negative control and Scl-Cre⁺ PTEN with constitutively activatedβ-catenin (Pten:Ctnnb1) double mutant bone marrow and spleen.

The absolute numbers (per femur+tibia) of lineage negative, Sca-1⁺ Kit⁺(LSK) cells in Scl-Cre negative control and Scl-Cre⁺ PTEN withconstitutively activated β-catenin (Pten:Ctnnb1) double mutant and eachsingle mutant bone marrow (FIG. 1A top) and spleen (FIG. 1A bottom) asdetermined by FACS analysis were counted. (Harada, N., et al., Embo J,18(21): 5931-42 1999. Yilmaz, O. H., et al., Nature, 441:475-82 2006.Zhang, J., et al., Nature, 441(7092): 518-22 2006.) Mice are at 10 dayspost-induction of Tamoxifen. Reduction of LSK cells in double mutantbone marrow with expansion in the spleen is indicative of mobilizationfrom bone marrow to spleen.

FACS analysis was performed on LSK cells from Scl-Cre negative controland Scl-Cre⁺ PTEN with constitutively activated 8-catenin (Pten:Ctnnb1)double mutant and each single mutant bone marrow and spleen (see FIGS.1B-E for representative examples of FACS analysis). Cells were pre-gatedon live, lineage negative cells.

Absolute number of LSK cells per femur and tibia in control, Pten, andPten:Ctnnb1 mutant bone marrow (FIG. 1F) and spleen (FIG. 1G) at 6 weekspost-induction were counted. While the percentage of LSKs is increasedin double mutants (see FIG. 1C), low cellularity of bone marrow fromdouble mutants yields only moderately increased absolute numberscompared to control.

The percentage of LSK cells which were Flk-2⁻ (indicating long-termreconstituting (LT)-HSGs) in control, Pten, and Pten:Ctnnb1 mutant bonemarrow at 6 weeks post-induction were calculated. (FIGS. 1H-I). Thepercent of LT-HSC cells in Pten:Ctnnb1 double mutant mice were greatlyincreased in comparison to the control and to Pten single mutant mice.Ctnnb1 single mutants were not significantly different from controls atthis time point (data not shown).

FACS analysis of CD45 in leukemic Pten:Ctnnb1 mutant bone marrow wasperformed. As shown in FIG. 1J, CD45 (high) blast crisis cells areindicated in the blue box of the left panel. In comparison, no blastcell population is observed in control or Ctnnb1 single mutants while aminor one was observed in 1 of 8 Pten single mutant mice at 6 weekspost-induction (data not shown).

LSK analysis of leukemic Pten:Ctnnb1 mutant mouse bone marrow was alsoperformed (FIG. 1J, right panel). Note the conversion to blast cells(lower left) with only a remnant LSK population (compare to FIG. 1C).

Early hematopoietic progenitors were sorted and analyzed by FACS incontrol, Pten, and Pten:Ctnnb1 mutant bone marrow (FIG. 1K). Absolutenumbers of CMPs; GMPs; MEPs; and CLPs were determined.

Together, this data demonstrates the phenotypic effect of the geneticloss of PTEN coupled with constitutive activation of β-catenin in HSCs.While loss of PTEN alone results in a slight but significant expansionin splenic HSCs due to mobilization from the bone marrow, double mutantHSCs exhibit the greatest mobilization at 10 days post-induction. By sixweeks post-induction, only double mutant splenic HSCs are dramaticallyincreased while single mutants are not significantly different fromcontrols. In addition, this dramatic increase in HSCs is not accompaniedby an increase in early hematopoietic progenitors; rather these earlyprogenitors are all reduced with the exception of CLPs which are notsignificantly different from control. HSCs accumulate dramatically inthe spleen of double, but not single, mutants by proliferation withreduced differentiation. Thus, surprisingly and unexpectedly, loss ofPTEN coupled with the constitutive activation of β-catenin drives stemcell self-renewal while neither pathway individually is capable ofdriving long-term self-renewal.

Example 2 In Vitro Culture of Control and Mutant LSK Cells Cell Culture

LSK or LSK Flk2⁻ cells were sorted into 96-well U-bottom tissue cultureplates at 100 cells/well with 200 μl media/well. Cells were incubated at37° C., 5% O₂, 5% CO₂ (balance N₂) for the indicated number of days.One-half total volume of media (see Table 1, below for the base media)was carefully pipetted from the top and replaced with fresh media everyother day.

TABLE 1 Base Media Components Source StemSpan Media: (Iscove's-modifiedDulbecco's Stem Cell medium (IMDM) supplemented with 1% bovineTechnologies; serum albumin, 10 μg ml⁻¹ recombinant human Cat. No. 09600insulin, 200 μg ml⁻¹ iron-saturated transferrin, 0.1 mM2-mercaptoethanol and 2 mM glutamine.) 10 μg/ml Heparin Sigma, Cat. No.H-3149 0.5X Penicillin/Streptomycin Sigma, Cat. No. P4333 10 ng/mlrecombinant mouse (rm) Stem Cell Factor Biovision, Cat. No. 4328-10 20ng/ml rm-Thrombopoietin Cell Sciences, Inc, Cat. No. CRT401B

Double Mutant HSCs Expand Dramatically In Vitro and In Vivo but Fail toDifferentiate.

For the following experiments, the base media from Table 1 was furthersupplemented with 20 ng/ml rm-IGF-2 (R&D Systems, Cat. No. 792-MG) and10 ng/ml recombinant human FGF-1 (Affinity BioReagents, Cat. No.ORP16010).

One hundred LSK cells isolated from control, constitutively active6-catenin (Ctnnb1), Pten mutant, and double mutant (Pten:Ctnnb1) micewere cultured for 10 days (FIG. 2A). Cell numbers were not dramaticallyincreased from the 100 seeded LSKs in control, while Ctnnb1 singlemutant LSKs did not survive. In contrast, Pten single mutant LSKsexhibited greater proliferation but appeared more heterogeneousindicating more significant differentiation. The greatest and mosthomogeneous expansion occurred from Pten:Ctnnb1 double mutant LSKs.

LSK cells isolated from Pten and Pten:Ctnnb1 mutants were examined at 34days culture (FIG. 2B). Wild-type control cultures did not expand beyond4 weeks; Ctnnb1 mutant cultures did not survive beyond 10 days. Ptenmutant HSC cultures appeared more heterogenous with significant cellclumping and more irregular cell morphology. Also note thespindle-shaped adherent cells (arrows) showing differentiation. Incontrast, double mutant HSC cultures exhibited consistent morphology.Therefore, while Pten single mutant LSKs survived and expanded, they hadundergone more significant differentiation than the much morehomogeneous Pten:Ctnnb1 double mutant LSKs.

At 7 weeks culture, Pten and Pten:Ctnnb1 LSK cultures were counted andanalyzed by FACS for maintenance of the LSK phenotype (FIG. 2E). Again,wild-type control and Ctnnb1 cultures did not survive this long invitro. Double mutant LSKs undergo >1,200 fold expansion compared to 50fold for Pten single mutant LSKs. LSK purity of cultures wassignificantly greater in Pten:Ctnnb1 cultures maintaining the LSKphenotype in about 85% of total live cells compared to about 50% forPten single mutant cultures.

Collectively these data demonstrate that double mutant HSCs can becultured longer and with far greater expansion than either single mutantor control HSCs.

Example 3 Transplantation Analysis of Pten and Pten:Ctnnb1 LSK Cellsafter 5 Weeks of Culture

For the following experiments, cells were cultured in the same manner asdescribed in Example 2. As in Example 2, the base media of Table 1 wassupplemented with 20 ng/ml rm-IGF-2 (R&D Systems, 792-MG) and 10 ng/mlrecombinant human FGF-1 (Affinity BioReagents, ORP16010).

At 5 weeks culture, Pten and Pten:Ctnnb1 LSK cultures were re-sorted and1000 LSK cells (CD45.2⁺) from each were transplanted into lethallyirradiated (10Gy) CD45.1⁺ recipient mice along with 2×10⁵ congenic wholebone marrow competitor cells. Because wild-type cells did not survive 5weeks culture, 1000 fresh wild-type LSK cells were also transplanted asa separate control group. At 4 weeks post-transplant, there was nodetectable engraftment from peripheral blood analysis of micetransplanted with either Pten or Pten:Ctnnb1 LSK cultures (data notshown). At 5 weeks post-transplant, bone marrow from recipient mice wasanalyzed for donor engraftment (CD45.2⁺ cells) and donor LSK cells(CD45.2⁺ LSKs).

FIGS. 2F and 2G show representative donor engraftment (left) and donorLSK cell engraftment (right) from bone marrow of mice transplanted with1000 fresh LSK cells (F) or 1000 cultured Pten:Ctnnb1 LSK cells (G).FIGS. 2H, 2I, and 2J show quantitation of donor (CD45.2⁺) cells (H),donor LSK cells (I), and fold-increase in donor LSKs (FIG. 2J) isolatedfrom bone marrow of recipient mice described in FIGS. 2F and G at 5weeks post-transplant. Although the absolute number of Pten:Ctnnb1 donorcells per fermur is lower than that of control or Pten single mutantdonor cells, the number of Pten:Ctnnb1 donor LSK cells is significantlyhigher than either that of control or Pten single mutant donor LSKcells.

Collectively, these data demonstrate that double mutant HSCs can becultured longer and with far greater expansion than either single mutantor control HSCs. However, permanent genetic alteration of both pathwaysleads to an increased ability to self-renew both in vitro as well as invivo following long-term culture but a failure to differentiate and thusrepopulate the hematopoietic system of transplant recipients. Thisfurther demonstrates the ability of the PTEN and β-catenin signalingpathways to cooperatively drive stem cell expansion by proliferationwithout differentiation

Example 4

Ex vivo pharmacological manipulation of the PTEN/Akt and Wnt/β-cateninsignaling pathways cooperatively drive functional HSC expansion

One hundred LSK Flk2⁻ cells were sorted from wild-type (C57Bl/6) miceand cultured in (1) media, (2) media+1 μM CHIR99021 (a GSK-3(3βinhibitor, a gift from Dr. Sheng Ding), (3) media+200 nM DipotassiumBis-peroxo(picolinato)oxovanadate (BpV(pic), a PTEN inhibitor, availablefrom Calbiochem, Cat. No. 203705), (4) media+1 μM CHIR99021+200 nMBpV(pic), (5) media+200 nM Shikonin (also a PTEN inhibitor, availablefrom Calbiochem, Cat. No. 565850), and (6) media+200 nM Shikonin +1 μMCHIR99021. (FIGS. 3B-C). Cells were cultured as described above. Cellswere examined at 17 days of culture (FIG. 3B, original magnification100×) and 23 days (FIG. 3C, original magnification 40×). Compared tocontrol, both inhibitors applied individually exhibited greaterexpansion of LSK cells indicating that GSK-3β inhibition is not strictlyequivalent to constitutive activation of β-catenin shown in Ctnnb1mutant LSKs, while BpV(pic) exhibited similar results compared to Ptenmutant LSKs (see FIG. 2). Similar to double mutant LSKs (FIG. 2), thegreatest expansion occurred with both inhibitors present (FIG. 3B/Cpanel 4).

LSK Flk2⁻ cells at 28 days culture in the indicated media conditionswere examined (FIG. 3D, original magnification 200×). Here, significantexpansion relative to control was observed with both inhibitors presentindividually; however, significant differentiation/heterogeneity of cellmorphology was observed in both cases, including more variable cellsize/morphology and/or differentiation to adherent, spindle-shaped cells(middle panels). In contrast, expansion with homogeneity was achievedwhen both inhibitors were present (last panel).

FACS analysis of 28 day LSK Flk2⁻ cells cultured inmedia+BpV(pic)+CHIR99021 (FIG. 3E) was performed. Cells were pre-gatedon live, lineage negative cells. Greater than 90% of LSKs retained Flk2negativity (data not shown). Thus, the LSK Flk2⁻ phenotype wasmaintained with high purity in cultures containing both inhibitors.

Fold expansion of LSK Flk2⁻ cells after 28 days culture in (1) media,(2) media+BpV(pic), (3) media+CHIR99021, and (4) media+CHIR99021+BpV(pic) were analyzed. While each inhibitor added individually led tosignificant expansion compared to media without either inhibitor, thegreatest expansion (−270 fold) was observed when both inhibitors wereadded together.

Collectively, these data demonstrate that the PTEN/Akt and Wnt/β-cateninsignaling pathways can be manipulated pharmacologically to drive HSCexpansion. The greatest expansion is achieved by manipulating bothpathways simultaneously. Functional, short-term HSCs show highestreconstitution ability when cultured in the presence of both inhibitors.Substantial longer-term reconstitution (8 weeks) occurs only when HSCsare cultured in the presence of both inhibitors but not when culturedwith either single inhibitor or in the absence of either inhibitor.Thus, the pharmacological manipulation of both pathways simultaneouslyresults in the greatest expansion of functional HSCs.

Example 5 Transplantation Analysis of Cultured LSK Cells after Ex VivoPharmacological Manipulation Cell Harvest and Repopulation

Cells were harvested from the wells prior to transplantation bypipetting up and down several times before transferring to a fresh tube.Residual was then collected by adding more media and repeating. Cellswere washed in DMEM (Invitrogen, Cat. No. 31053) without phenol red andadded to the appropriate number of whole bone marrow rescue cells from acongenic donor (for 200,000 rescue cells +1,000 re-sorted LSK Flk2⁻cells (FIG. 3F-H) or the non-adherent product of 10 days culture of 100LSK Flk2⁻ cultured cells (FIG. 3I-K) per mouse as indicated). Cells wereinjected into lethally irradiated (10 Grays, single dose) Ptprc(CD45.1⁺) recipient mice through the tail vein using an insulin syringe.

Repopulation was measured at 4 weeks post-transplant by collection ofperiperal blood, red blood cell lysis, and staining of CD45.1(recipient) compared to CD45.2 (donor) engraftment using antibodiespurchased from eBiosciences (FITC conjugated CD45.2 (Cat. No.11-0454-85) and PE-Cy5 conjugated CD45.1 (Cat. No. 15-0453-82)). Micetransplanted with rescue/competitor cells only were used as a control todetermine the limits of repopulation detection. Multi-lineagereconstitution was determined by CD3, B220 (for lymphoid) and Gr1, Mac-1(for myeloid), as described above.

Transplantation Analysis of 28 Day Cultures.

Cells cultured for 28 days in (1) media, (2) media+BpV(pic), (3) media+CHIR99021 and (4) media+CHIR99021 (1 μM)+BpV(pic) (200 nM) werere-sorted for LSK Flk2⁻ cells. One thousand LSK Flk2⁻ cells (CD45.2⁺)from each media condition were transplanted into lethally irradiated(10Gy) CD45.1⁺ recipient mice along with 2×10⁵ congenic whole bonemarrow competitor cells. At 4 weeks post-transplant, peripheral bloodwas analyzed for donor (FIG. 3G) and multi-lineage (FIG. 3H)engraftment. In FIG. 3G, each bar represents an individual mouse. Thehorizontal-dashed line represents the average ‘engraftment’ of micetransplanted with competitor cells only and, thus, the limit ofdetectability for true engraftment. Long-term (4 month) engraftment hasnot been observed from 28-day cultures (data not shown). Six of 8 miceshow >1% engraftment when transplanted with LSK Flk2⁻ cells culturedwith both inhibitors present compared to 4/8 with only CHIR99021present, 0/10 with only BpV(pic) present, and 2/6 with media only. Onepercent or greater engraftment is a standard limit for substantialengraftment. (Zhang, C. C., et al., Nat Med, 12(2): 240-5, 2006. Zhang,C.C. and H.F. Lodish, Blood, 105(11): 4314-20, 2005). Thus, while bothinhibitors together leads to greatest expansion in LSKs (FIG. 2F),transplantation of equivalent numbers of these cultured LSK Flk2⁻ cellsalso leads to increased short-term engraftment/functionality whencultured with both inhibitors compared to no or either single inhibitoronly.

While all mice with genetic alterations resulting in constitutivelyactive Jβ-catenin and loss of PTEN will develop leukemia and must besacrificed due to poor health within 8-10 weeks post-mutation induction(FIG. 11 and data not shown), no mice transplanted with LSK Flk2⁻ cellscultured in either inhibitor singly or in combination has shown any signof leukogenesis up to 16 weeks post-transplantation. All such miceappeared healthy unlike 8-10 weeks post-induction genetically doublemutant mice, exhibiting no loss of body weight, anemia, loss ofappetite, lethargy, hunched posture, etc. Thus, the effects of theinhibition of both pathways using, e.g., BpV(pic) and CHIR99021, isreversible.

Transplantation Analysis of 10 Day Cultures.

Cells cultured for 9 days in (1) media, (2) media+BpV(pic) (200 nM), (3)media+CHIR99021 (100 nM), and (4) media+CHIR99021 (100 nM)+BpV(pic) (200nM) were re-sorted for LSK Flk2⁻ cells, and fold expansion of LSK Flk2⁻cells after 9 days culture in the indicated conditions was determined(FIG. 3I). Because long-term engraftment was not observed from 28 daycultures (FIG. 3D-H and data not shown), LSK Flk2⁻ cells were culturedfor only 9 days to test if both expansion and long-term repopulationcould be achieved. Similar trends were observed here when compare to the28 day cultures (compare to FIG. 9F) although the extent of expansionwas substantially reduced at only 9 days versus 28 days culture.

FACS analysis was performed on 9 day LSK Flk2⁻ cells cultured inmedia+BpV(pic) (200 nM)+CHIR99021 (100 nM) (FIG. 3J). Cells werepre-gated on live, lineage negative cells. Greater than 90% of LSKsretain Flk2 negativity (data not shown). Here, the levels of Sca-1 andKit appear normal compared to the Sca-1^((high))Kit^((high)) populationshown from 28 day cultures (FIG. 1E).

Ten day cultures were transplanted into lethally irradiated (10Gy)CD45.1⁺ recipient mice along with 2×10⁵ congenic whole bone marrowcompetitor cells. The total, non-adherent cell product after 10 daysculture of 100 initial LSK Flk-2 cells was transplanted per mouse. At 8weeks post-transplant, peripheral blood was analyzed for donor (FIG. 3G)and multi-lineage (FIG. 3H) engraftment. As shown, multi-lineagereconstitution was observed from all mice exhibiting true engraftment(data not shown). In FIG. 3G, each bar represents an individual mouse;the horizontal-dashed line represents the average ‘engraftment’ of micetransplanted with competitor cells only and thus the limit ofdetectability for true engraftment. Here, 3/7 mice transplanted with LSKFlk2⁻ cells cultured in the presence of both inhibitors exhibited 1% orgreater donor engraftment compared to no mice reaching this threshold inthe single or no inhibitor groups.

Collectively, these data demonstrate that the PTEN/Akt and Wnt/6-cateninsignaling pathways can be manipulated pharmacologically to drive HSCexpansion. The greatest expansion is achieved only by manipulating bothpathways simultaneously. Functional, short-term HSCs show highestreconstitution ability when cultured in the presence of both inhibitors.Substantial longer-term reconstitution (8 weeks) occurs only when HSCsare cultured in the presence of both inhibitors but not when culturedwith either single inhibitor or in the absence of either inhibitor.Thus, the pharmacological manipulation of both pathways simultaneouslyresults in the greatest expansion of functional HSCs. This effect isreversible because recipient animals did not develop leukemia as geneticmutants did (FIG. 1) and cultured HSCs were able to differentiate unlikecultured HSCs from genetic mutants (FIG. 2).

Example 6 Culturing of HSC in Media Containing Biologics

Anti-GSK-3β and anti-PTEN antibodies may be made in accordance withprocedures known in the art (or purchased, e.g., from Sigma,ExactAntigene, and Biocompare).

One hundred LSK Flk2⁻ cells are sorted from wild-type (C57BI/6) mice andare cultured in (1) media, (2) media+an GSK-3β antibody, (3) media+ananti-PTEN antibody, and (4) media+anti-GSK-3β and anti-PTEN antibodies.Cells are cultured as described above. Cells are examined at 9 days, 17days and 23 days of culture. The greatest expansion of HSCs is expectedto occur when both antibodies are present.

Example 7 Culturing of HSC in Media Containing siRNA or RNAi

PTEN siRNA and GSK-3b siRNA may be made in accordance with proceduresknown in the art. (See, e.g., Mise-Omata S et al. Biochem Biophys ResCommun. 328(4):1034-42 2005, or may be purchased from Biocompare).

One hundred LSK Flk2⁻ cells are sorted from wild-type (C57BI/6) mice andare cultured in (1) media, (2) media+GSK-3β siRNA, (3) media+PTEN siRNA,and (4) media+GSK-3β siRNA and PTEN siRNA. Cells are cultured asdescribed above. Cells are examined at 9 days, 17 days and 23 days ofculture. The greatest expansion of HSC is expected to occur when bothsiRNAs are present.

All documents cited in this application are hereby incorporated byreference as if recited in full herein.

Although illustrative embodiments of the present invention have beendescribed herein, it should be understood that the invention is notlimited to those described, and that various other changes ormodifications may be made by one skilled in the art without departingfrom the scope or spirit of the invention.

What is claimed is: 1-40. (canceled)
 41. A kit for expanding anhematopoietic stem cell (HSC) population for subsequent transplantationinto a patient in need thereof, the kit comprising a PTEN inhibitor, aGSK-3β inhibitor, and instructions for the use of the inhibitors. 42.The kit according to claim 41, wherein the PTEN inhibitor is selectedfrom the group consisting of shikonin, a bisperoxovanadium compound,SF-1751 (Semafore Pharmaceuticals), pharmaceutical salts thereof, andcombinations thereof, and the GSK-3β inhibitor is selected from thegroup consisting of Hymenialdisine, Flavopiridol, Kenpaullone,Alsterpaullone, Azakenpaullone, Indirubin-30-oxime,6-Bromoindirubin-30-oxime (B10), 6-Bromoindirubin-30-acetoxime, AloisineA, Aloisine B, TDZD8, Compound 12, CHIR98014, CHIR99021 (CT99021),CT20026, Compound 1, SU9516, ARA014418, Staurosporine, Compound 5a,Compound 29, Compound 46, GF109203x (bisindolylmaleimide I), Ro318220(bisindolylmaleimide IX), SB216763, SB415286, 15, CGP60474, Compound 8b,TWS119, Compound 1A, Compound 17, Lithium, Beryllium, Zinc, smallmolecule GSK-3β inhibitors (Vertex Pharmaceuticals), NP-12(Neuropharma), GSK-3β inhibitors (Amphora), GSK-3β inhibitors(CrystalGenomics), SAR-502250 (Sanofi-Aventis), 3544 (Hoffmann-LaRoche), GSK-3β inhibitors (Lundbeck), TDZD-8 (Cancer Center, Universityof Rochester), pharmaceutically acceptable salts thereof, andcombinations thereof.
 43. The kit according to claim 42, wherein thebisperoxovanadium compound is selected from the group consisting ofbpV(phen)2, bpV(pic), pharmaceutical salts thereof, and combinationsthereof.
 44. The kit according to claim 41, wherein the PTEN inhibitoris bpV(pic), and the GSK-3β inhibitor is CHIR99201. 45-96. (canceled)97. The kit according to claim 41, wherein the HSC is obtained from amammalian tissue selected from the group consisting of cord blood,peripheral blood, and bone marrow.
 98. The kit according to claim 41,wherein the PTEN inhibitor is reversible.
 99. The kit according to claim98, wherein the reversible PTEN inhibitor is selected from the groupconsisting of shikonin, a bisperoxovanadium compound, SF-1751 (SemaforePharmaceuticals), pharmaceutical salts thereof, and combinationsthereof.
 100. The kit according to claim 99, wherein thebisperoxovanadium compound is selected from the group consisting ofbpV(phen)₂, bpV(pic), pharmaceutical salts thereof, and combinationsthereof.
 101. The kit according to claim 41, wherein the GSK-3βinhibitor is reversible.
 102. The kit according to claim 101, whereinthe reversible GSK-3β inhibitor is selected from the group consisting ofHymenialdisine, Flavopiridol, Kenpaullone, Alsterpaullone,Azakenpaullone, Indirubin-30-oxime, 6-Bromoindirubin-30-oxime (B10),6-Bromoindirubin-30-acetoxime, Aloisine A, Aloisine B, TDZD8, Compound12, CHIR98014, CHIR99021 (CT99021), CT20026, Compound 1, SU9516,ARA014418, Staurosporine, Compound 5a, Compound 29, Compound 46,GF109203x (bisindolylmaleimide I), Ro318220 (bisindolylmaleimide IX),SB216763, SB415286, 15, CGP60474, Compound 8b, TWS119, Compound 1A,Compound 17, Lithium, Beryllium, Zinc, small molecule GSK-3β inhibitors(Vertex Pharmaceuticals), NP-12 (Neuropharma), GSK-3β inhibitors(Amphora), GSK-3β inhibitors (CrystalGenomics), SAR-502250(Sanofi-Aventis), 3544 (Hoffmann-La Roche), GSK-3β inhibitors(Lundbeck), TDZD-8 (Cancer Center, University of Rochester),pharmaceutically acceptable salts thereof, and combinations thereof.103. The kit according to claim 102, wherein the reversible GSK-3βinhibitor is CHIR99021.